Targeted conjugates and particles and formulations thereof

ABSTRACT

Nanoparticles and microparticles, and pharmaceutical formulations thereof, containing conjugates of an active agent such as a therapeutic, prophylactic, or diagnostic agent attached to a targeting moiety, such as a somatostatin receptor binding moiety, via a linker have been designed. Such nanoparticles and microparticles can provide improved temporospatial delivery of the active agent and/or improved biodistribution. Methods of making the conjugates, the particles, and the formulations thereof are provided. Methods of administering the formulations to a subject in need thereof are provided, for example, to treat or prevent cancer or infectious diseases.

REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 15/652,947 filed Jul. 18, 2017, entitled TARGETED CONJUGATESAND PARTICLES AND FORMULATIONS THEREOF, which is a continuationapplication of U.S. application Ser. No. 15/382,487 filed Dec. 16, 2016,entitled TARGETED CONJUGATES AND PARTICLES AND FORMULATIONS THEREOF,which is a continuation application of PCT application No.PCT/US2015/038569 filed Jun. 30, 2015, entitled TARGETED CONJUGATES ANDPARTICLES AND FORMULATIONS THEREOF, which claims priority to U.S.Provisional Patent Application No. 62/019,001 filed Jun. 30, 2014,entitled TARGETED CONJUGATES ENCAPSULATED IN PARTICLES AND FORMULATIONSTHEREOF, U.S. Provisional Patent Application No. 62/077,487 filed Nov.10, 2014, entitled TARGETED CONJUGATES ENCAPSULATED IN PARTICLES ANDFORMULATIONS THEREOF and U.S. Provisional Patent Application No.62/150,413 filed Apr. 21, 2015, entitled TARGETED CONJUGATES ANDPARTICLES AND FORMULATIONS THEREOF, the contents of each of which areherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention generally relates to the field of targeting ligands,conjugates thereof, and particles for drug delivery. More particularly,the invention relates to the use of molecules targeting somatostatinreceptors, e.g., for treating cancer.

BACKGROUND OF THE INVENTION

Developments in nanomedicine are generally directed towards improvingthe pharmaceutical properties of the drugs and, in some cases, enhancingthe targeted delivery in a more cell-specific manner. Severalcell-specific drugs have been described, and include monoclonalantibodies, aptamers, peptides, and small molecules. Despite some of thepotential advantages of such drugs, a number of problems have limitedtheir clinical application, including size, stability, manufacturingcost, immunogenicity, poor pharmacokinetics and other factors.

Nanoparticulate drug delivery systems are attractive for systemic drugdelivery because they may be able to prolong the half-life of a drug incirculation, reduce non-specific uptake of a drug, and improveaccumulation of a drug at tumors, e.g., through an enhanced permeationand retention (EPR) effect. There are limited examples of therapeuticsformulated for delivery as nanoparticles, which include DOXIL®(liposomal encapsulated doxorubicin) and ABRAXANE® (albumin boundpaclitaxel nanoparticles).

The development of nanotechnologies for effective delivery of drugs ordrug candidates to specific diseased cells and tissues, e.g., to cancercells, in specific organs or tissues, in a temporospatially regulatedmanner potentially can overcome or ameliorate therapeutic challenges,such as systemic toxicity. However, while targeting of the deliverysystem may preferentially deliver drug to a site where therapy isneeded, the drug released from the nanoparticle may not for example,remain in the region of the targeted cells in efficacious amounts or maynot remain in the circulation in a relatively non-toxic state for asufficient amount of time to decrease the frequency of treatment orpermit a lower amount of drug to be administered while still achieving atherapeutic effect. Accordingly, there is a need in the art for improveddrug targeting and delivery, including identification of targetingmolecules that can be incorporated into particles and whose presencedoes not substantially interfere with efficacy of the drug.

SUMMARY OF THE INVENTION

Applicants have created molecules that are conjugates of a somatostatinreceptor binding moiety and an active agent, e.g., a cancer therapeuticagent such as a platinum-containing agent. Furthermore, such conjugatescan be encapsulated into particles. The conjugates and particles areuseful for delivering active agents such as tumor cytotoxic agents tocells expressing somatostatin receptors (SSTRs).

Applicants have developed novel conjugates and particles, includingpolymeric nanoparticles, and pharmaceutical formulations thereof. Theconjugates of an active agent such as a therapeutic, prophylactic, ordiagnostic agent are attached via a linker to a targeting moiety thatcan bind a somatostatin receptor. The conjugates and particles canprovide improved temporospatial delivery of the active agent and/orimproved biodistribution compared to delivery of the active agent alone.In some cases, the targeting moiety can also act as a therapeutic agent.In some embodiments, the targeting agent does not substantiallyinterfere with efficacy of the therapeutic agent in vivo. Methods ofmaking conjugates, particles, and formulations comprising such particlesare described herein. Such particles are useful for treating orpreventing diseases that are susceptible to the active agent, forexample, treating or preventing cancer or infectious diseases.

The conjugates include a targeting ligand and an active agent connectedby a linker, wherein the conjugate in some embodiments has the formula:(X—Y—Z)

wherein X is a somatostatin receptor targeting moiety; Y is a linker;and Z is an active agent.

One ligand can be conjugated to two or more active agents where theconjugate has the formula: X—(Y—Z)_(n). In other embodiments, one activeagent molecule can be linked to two or more ligands wherein theconjugate has the formula: (X—Y)_(n)—Z. n is an integer equal to orgreater than 1.

The targeting moiety, X, can be any somatostatin receptor binding moietysuch as, but not limited to, somatostatin, octreotide, octreotate,vapreotide, pasireotide, lanreotide, seglitide, or any other example ofsomatostatin receptor binding ligands. In some embodiments, thetargeting moiety is a somatostatin receptor binding moiety that binds tosomatostatin receptors 2 and/or 5.

The linker, Y, is bound to one or more active agents and one or moretargeting ligands to form a conjugate. The linker Y is attached to thetargeting moiety X and the active agent Z by functional groupsindependently selected from an ester bond, disulfide, amide,acylhydrazone, ether, carbamate, carbonate, and urea. Alternatively thelinker can be attached to either the targeting ligand or the active drugby a non-cleavable group such as provided by the conjugation between athiol and a maleimide, an azide and an alkyne. The linker isindependently selected from the group consisting alkyl, cycloalkyl,heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally issubstituted with one or more groups, each independently selected fromhalogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy,aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl,arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of thecarboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate,alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, orheterocyclyl is optionally substituted with one or more groups, eachindependently selected from halogen, cyano, nitro, hydroxyl, carboxyl,carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl,alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl.

In some embodiments, the linker comprises a cleavable functionality. Thecleavable functionality may be hydrolyzed in vivo or may be designed tobe hydrolyzed enzymatically, for example by Cathepsin B.

The active agent, Z, also referred as a payload, can be a therapeutic,prophylactic, diagnostic, or nutritional agent. In some embodiments, theactive agent, Z, may be an anti-cancer agent, chemotherapeutic agent,antimicrobial, anti-inflammatory agent, or combination thereof.

In some embodiments, the conjugate can be a compound according toFormula Ia:

wherein X is a somatostatin receptor targeting moiety defined above; Zis an active agent; X′, R¹, Y′, R² and Z′ are as defined herein.

X′ is either absent or independently selected from carbonyl, amide,urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or morenatural or unnatural amino acids, thio or succinimido; R¹ and R² areeither absent or comprised of alkyl, substituted alkyl, aryl,substituted aryl, polyethylene glycol (2-30 units); Y′ is absent,substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol(2-30 units) or an amide; Z′ is either absent or independently selectedfrom carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy,arylamino, thio or succinimido. In some embodiments, the linker canallow one active agent molecule to be linked to two or more targetingligands, or one targeting ligand to be linked to two or more activeagents.

In some embodiments, the conjugate can be a compound where linker Y isA_(m) according to Formula Ib:

wherein A is defined herein, m=0-20.

A in Formula Ia is a spacer unit, either absent or independentlyselected from the following substituents. For each substituent, thedashed lines represent substitution sites with X, Z or anotherindependently selected unit of A wherein the X, Z, or A can be attachedon either side of the substituent:

wherein z=0-40, R is H or an optionally substituted alkyl group, and R′is any side chain found in either natural or unnatural amino acids.

In some embodiments, the linker can be a compound according to FormulaIc:

wherein A is defined above, m=0-40, n=0-40, x=1-5, y=1-5, and C is abranching element defined herein.

C in Formula Ic is a branched unit containing three to sixfunctionalities for covalently attaching spacer units, ligands, oractive drugs, selected from amines, carboxylic acids, thiols, orsuccinimides, including amino acids such as lysine, 2,3-diaminopropanoicacid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, andcysteine.

A non-limiting example of a conjugate of the invention is a compoundselected from the group consisting of the following compounds:

In some embodiments, the active agent Z is DM1. In some embodiments, thesomatostatin receptor targeting moiety X is selected from somatostatin,seglitide, Tyr³-octreotate (TATE), cyclo(AA-Tyr-DTrp-Lys-Thr-Phe), oranalogs or derivatives thereof. X may covalently bind to linker Y at itsC-terminus or N-terminus. In some embodiments, the targeting moiety Xcomprises at least one D-Phe residue and the phenyl ring of the D-Pheresidue of the targeting moiety X has been replaced by alinker-containing moiety.

In one aspect, hydrophobic ion-pairing complexes containing theconjugate of the invention and counterions are provided. In someembodiments, the counterions are negatively charged. In another aspect,particles containing the conjugate of the invention or the hydrophobicion-pairing complexes of the conjugate of the invention are provided. Inanother aspect, pharmaceutical formulations are provided containing theconjugates or particles containing the conjugates described herein, orpharmaceutically acceptable salts thereof, in a pharmaceuticallyacceptable vehicle.

In one aspect, particles containing the conjugate of the invention areprovided. In some embodiments, the particle has a diameter between 10 nmand 5000 nm. In some embodiments, the particle has a diameter between 30nm and 70 nm, 120 nm and 200 nm, 200 nm and 5000 nm, or 500 nm-1000 nm.

Methods of making the conjugates and particles containing the conjugatesare provided. Methods are also provided for treating a disease orcondition, the method comprising administering a therapeuticallyeffective amount of the particles containing a conjugate to a subject inneed thereof. In an embodiment, the conjugates are targeted to a canceror hyperproliferative disease, for example, lymphoma, renal cellcarcinoma, leukemia, prostate cancer, lung cancer (e.g., small cell lungcancer (SCLC) and non-SCLC), pancreatic cancer (e.g., ductal), melanoma,colorectal cancer, ovarian cancer (e.g., epithelial ovarian cancer),breast cancer, glioblastoma (e.g., astrocytoma and glioblastomamultiforme), stomach cancer, liver cancer, sarcoma, bladder cancer,testicular cancer, esophageal cancer, head and neck cancer, endometrialcancer and leptomeningeal carcinomatosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the blood plasma concentration (μM) of theoctreotide-cabazitaxel conjugate of Example 1 as a function of time(hours) after tail vein injection in rats. The formulations injectedcontained either the free octreotide-cabazitaxel conjugate oroctreotide-cabazitaxel nanoparticles of Example 11.

FIG. 2 is a graph of the blood plasma concentration (μM) of theoctreotide-doxorubicin conjugate of Example 2 as a function of time(hours) after tail vein injection in rats. The formulations injectedcontained either the free octreotide-doxorubicin conjugate oroctreotide-doxorubicinnano particles of Example 12.

FIG. 3 is a graph of various conjugates represented as bars and showingon the Y-axis their activity in an H524 proliferation assay with andwithout competition by octreotide. The Y-axis shows the ratio of theIC₅₀ with octreotide added to the IC₅₀ without octreotide added. Thisassay demonstrates the extent to which the activity of the conjugatesdepends on the somatostatin receptors. Only DM1 conjugates show a ratiosignificantly greater than 1. This illustrates the surprising findingthat only DM1 conjugates show activity that is dependent on thereceptor.

FIG. 4 shows the tumor volume change over a period of up to 100 daysafter treatment with Conjugate 10, Conjugate 10 NP6 and DM1 in a NCI-H69model.

FIG. 5 is a graph of percent of mice with tumor <2000 mm³ over a periodof up to 100 days after treatment with Conjugate 10, Conjugate 10 NP6and DM1 in a NCI-H69 model.

FIG. 6 demonstrates tumor volume change over a period of 30 days withmultidoses of conjugate 10 and conjugate 10 NP6.

FIG. 7 shows rat plasma pK of conjugate 10 and conjugate 10 NP6.

FIG. 8 shows phospho-histone H3 response in NCI-H69 tumors aftertreatment with conjugate 10 and conjugate 10 NP6.

DETAILED DESCRIPTION OF THE INVENTION

At least five somatostatin receptors subtypes have been characterized,and tumors can express various receptor subtypes. (e.g., see Shaer etal., Int. 3. Cancer 70:530-537, 1997). Naturally occurring somatostatinand its analogs exhibit differential binding to receptor subtypes.Applicants have exploited this feature to create novel particles toimprove targeting of a conjugate comprising an active agent to a diseasetissue target. Such targeting can, for example, improve the amount ofactive agent at a site and decrease active agent toxicity to thesubject. As used herein, “toxicity” refers to the capacity of asubstance or composition to be harmful or poisonous to a cell, tissueorganism or cellular environment. Low toxicity refers to a reducedcapacity of a substance or composition to be harmful or poisonous to acell, tissue organism or cellular environment. Such reduced or lowtoxicity may be relative to a standard measure, relative to a treatmentor relative to the absence of a treatment.

Toxicity may further be measured relative to a subject's weight loss.where weight loss over 15%, over 20% or over 30% of the body weight isindicative of toxicity. Other metrics of toxicity may also be measuredsuch as patient presentation metrics including lethargy and generalmalaise. Neutropenia or thrombopenia may also be metrics of toxicity.

Pharmacologic indicators of toxicity include elevated AST/ALT levels,neurotoxicity, kidney damage, GI damage and the like.

The conjugates are released after administration of the particles. Thetargeted drug conjugates utilize active molecular targeting incombination with enhanced permeability and retention effect (EPR) andimproved overall biodistribution of the particles to provide greaterefficacy and tolerability as compared to administration of targetedparticles or encapsulated untargeted drug.

In addition, the toxicity of a conjugate containing a somatostatintargeting moiety linked to an active agent for cells that do not expressSSTRs is predicted to be decreased compared to the toxicity of theactive agent alone. Without committing to any particular theory,applicants believe that this feature is because the ability of theconjugated active agent to enter a cell is decreased compared theability to enter a cell of the active agent alone. Accordingly, theconjugates comprising an active agent and particles containing theconjugates as described herein generally have decreased toxicity fornon-SSTR expressing cells and at least the same or increased toxicityfor SSTR expressing cells compared to the active agent alone.

It is an object of the invention to provide improved compounds,compositions, and formulations for temporospatial drug delivery.

It is further an object of the invention to provide methods of makingimproved compounds, compositions, and formulations for temporospatialdrug delivery.

It is also an object of the invention to provide methods ofadministering the improved compounds, compositions, and formulations toindividuals in need thereof.

I. Definitions

The term “compound”, as used herein, is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. In the present application, compound is usedinterchangeably with conjugate. Therefore, conjugate, as used herein, isalso meant to include all stereoisomers, geometric isomers, tautomers,and isotopes of the structures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

The terms “subject” or “patient”, as used herein, refer to any organismto which the particles may be administered, e.g., for experimental,therapeutic, diagnostic, and/or prophylactic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, guinea pigs,cattle, pigs, sheep, horses, dogs, cats, hamsters, lamas, non-humanprimates, and humans).

The terms “treating” or “preventing”, as used herein, can includepreventing a disease, disorder or condition from occurring in an animalthat may be predisposed to the disease, disorder and/or condition buthas not yet been diagnosed as having the disease, disorder or condition;inhibiting the disease, disorder or condition, e.g., impeding itsprogress; and relieving the disease, disorder, or condition, e.g.,causing regression of the disease, disorder and/or condition. Treatingthe disease, disorder, or condition can include ameliorating at leastone symptom of the particular disease, disorder, or condition, even ifthe underlying pathophysiology is not affected, such as treating thepain of a subject by administration of an analgesic agent even thoughsuch agent does not treat the cause of the pain.

A “target”, as used herein, shall mean a site to which targetedconstructs bind. A target may be either in vivo or in vitro. In certainembodiments, a target may be cancer cells found in leukemias or tumors(e.g., tumors of the brain, lung (small cell and non-small cell), ovary,prostate, breast and colon as well as other carcinomas and sarcomas). Instill other embodiments, a target may refer to a molecular structure towhich a targeting moiety or ligand binds, such as a hapten, epitope,receptor, dsDNA fragment, carbohydrate or enzyme. A target may be a typeof tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue,liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.

The “target cells” that may serve as the target for the method orconjugates or particles, are generally animal cells, e.g., mammaliancells. The present method may be used to modify cellular function ofliving cells in vitro, i.e., in cell culture, or in vivo, in which thecells form part of or otherwise exist in animal tissue. Thus, the targetcells may include, for example, the blood, lymph tissue, cells liningthe alimentary canal, such as the oral and pharyngeal mucosa, cellsforming the villi of the small intestine, cells lining the largeintestine, cells lining the respiratory system (nasal passages/lungs) ofan animal (which may be contacted by inhalation of the subjectinvention), dermal/epidermal cells, cells of the vagina and rectum,cells of internal organs including cells of the placenta and theso-called blood/brain barrier, etc. In general, a target cell expressesat least one type of SSTR. In some embodiments, a target cell can be acell that expresses an SSTR and is targeted by a conjugate describedherein, and is near a cell that is affected by release of the activeagent of the conjugate. For example, a blood vessel expressing an SSTRthat is in proximity to a tumor may be the target, while the activeagent released at the site will affect the tumor.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The term thusmeans any substance intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease, disorder or condition in theenhancement of desirable physical or mental development and conditionsin an animal, e.g., a human.

The term “modulation” is art-recognized and refers to up regulation(i.e., activation or stimulation), down regulation (i.e., inhibition orsuppression) of a response, or the two in combination or apart. Themodulation is generally compared to a baseline or reference that can beinternal or external to the treated entity.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract (enteral) or non-invasivetopical routes. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraperitoneally, intrapleurally, intratracheally, intraossiously,intracerebrally, intrathecally, intramuscularly, subcutaneously,subjunctivally, by injection, and by infusion.

“Topical administration”, as used herein, means the non-invasiveadministration to the skin, orifices, or mucosa. Topical administrationcan be delivered locally, i.e., the therapeutic can provide a localeffect in the region of delivery without systemic exposure or withminimal systemic exposure. Some topical formulations can provide asystemic effect, e.g., via adsorption into the blood stream of theindividual. Topical administration can include, but is not limited to,cutaneous and transdermal administration, buccal administration,intranasal administration, intravaginal administration, intravesicaladministration, ophthalmic administration, and rectal administration.

“Enteral administration”, as used herein, means administration viaabsorption through the gastrointestinal tract. Enteral administrationcan include oral and sublingual administration, gastric administration,or rectal administration.

“Pulmonary administration”, as used herein, means administration intothe lungs by inhalation or endotracheal administration. As used herein,the term “inhalation” refers to intake of air to the alveoli. The intakeof air can occur through the mouth or nose.

The terms “sufficient” and “effective”, as used interchangeably herein,refer to an amount (e.g., mass, volume, dosage, concentration, and/ortime period) needed to achieve one or more desired result(s). A“therapeutically effective amount” is at least the minimum concentrationrequired to affect a measurable improvement or prevention of at leastone symptom or a particular condition or disorder, to affect ameasurable enhancement of life expectancy, or to generally improvepatient quality of life. The therapeutically effective amount is thusdependent upon the specific biologically active molecule and thespecific condition or disorder to be treated. Therapeutically effectiveamounts of many active agents, such as antibodies, are known in the art.The therapeutically effective amounts of compounds and compositionsdescribed herein, e.g., for treating specific disorders may bedetermined by techniques that are well within the craft of a skilledartisan, such as a physician.

The terms “bioactive agent” and “active agent”, as used interchangeablyherein, include, without limitation, physiologically orpharmacologically active substances that act locally or systemically inthe body. A bioactive agent is a substance used for the treatment (e.g.,therapeutic agent), prevention (e.g., prophylactic agent), diagnosis(e.g., diagnostic agent), cure or mitigation of disease or illness, asubstance which affects the structure or function of the body, orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

The term “prodrug” refers to an agent, including a small organicmolecule, peptide, nucleic acid or protein, that is converted into abiologically active form in vitro and/or in vivo. Prodrugs can be usefulbecause, in some situations, they may be easier to administer than theparent compound (the active compound). For example, a prodrug may bebioavailable by oral administration whereas the parent compound is not.The prodrug may also have improved solubility in pharmaceuticalcompositions compared to the parent drug. A prodrug may also be lesstoxic than the parent. A prodrug may be converted into the parent drugby various mechanisms, including enzymatic processes and metabolichydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed.Progress in Drug Research, 4:221-294; Morozowich et al. (1977)Application of Physical Organic Principles to Prodrug Design in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977) BioreversibleCarriers in Drug in Drug Design, Theory and Application, APhA; H.Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999)Prodrug approaches to the improved delivery of peptide drug, Curr.Pharm. Design. 5(4):265-287; Pauletti et al. (1997) Improvement inpeptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Estersas Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech.11:345-365; Gaignault et al. (1996) Designing Prodrugs and BioprecursorsI. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asgharnejad (2000).Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Leeand E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems,Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for theimprovement of drug absorption via different routes of administration,Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53; Balimane and Sinko(1999). Involvement of multiple transporters in the oral absorption ofnucleoside analogues, Adv. Drug Delivery Rev., 39(1-3): 183-209; Browne(1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol. 20(1): 1-12;Bundgaard (1979). Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs, Arch. Pharm.Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985) Design of Prodrugs, NewYork: Elsevier; Fleisher et al. (1996) Improved oral drug delivery:solubility limitations overcome by the use of prodrugs, Adv. DrugDelivery Rev. 19(2): 115-130; Fleisher et al. (1985) Design of prodrugsfor improved gastrointestinal absorption by intestinal enzyme targeting,Methods Enzymol. 112: 360-81; Farquhar D, et al. (1983) BiologicallyReversible Phosphate-Protective Groups, J. Pharm. Sci., 72(3): 324-325;Han, H. K. et al. (2000) Targeted prodrug design to optimize drugdelivery, AAPS PharmSci., 2(1): E6; Sadzuka Y. (2000) Effective prodrugliposome and conversion to active metabolite, Curr. Drug Metab.,1(1):31-48; D. M. Lambert (2000) Rationale and applications of lipids asprodrug carriers, Eur. J. Pharm. Sci., 11 Suppl. 2:S15-27; Wang, W. etal. (1999) Prodrug approaches to the improved delivery of peptide drugs.Curr. Pharm. Des., 5(4):265-87.

The term “biocompatible”, as used herein, refers to a material thatalong with any metabolites or degradation products thereof that aregenerally non-toxic to the recipient and do not cause any significantadverse effects to the recipient. Generally speaking, biocompatiblematerials are materials which do not elicit a significant inflammatoryor immune response when administered to a patient.

The term “biodegradable” as used herein, generally refers to a materialthat will degrade or erode under physiologic conditions to smaller unitsor chemical species that are capable of being metabolized, eliminated,or excreted by the subject. The degradation time is a function ofcomposition and morphology. Degradation times can be from hours toweeks.

The term “pharmaceutically acceptable”, as used herein, refers tocompounds, materials, compositions, and/or dosage forms that are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio, in accordance withthe guidelines of agencies such as the U.S. Food and DrugAdministration. A “pharmaceutically acceptable carrier”, as used herein,refers to all components of a pharmaceutical formulation that facilitatethe delivery of the composition in vivo. Pharmaceutically acceptablecarriers include, but are not limited to, diluents, preservatives,binders, lubricants, disintegrators, swelling agents, fillers,stabilizers, and combinations thereof.

The term “molecular weight”, as used herein, generally refers to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (Mw) as opposed to the number-average molecular weight(Me). Capillary viscometry provides estimates of molecular weight as theinherent viscosity determined from a dilute polymer solution using aparticular set of concentration, temperature, and solvent conditions.

The term “small molecule”, as used herein, generally refers to anorganic molecule that is less than 2000 g/mol in molecular weight, lessthan 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.

The term “hydrophilic”, as used herein, refers to substances that havestrongly polar groups that readily interact with water.

The term “hydrophobic”, as used herein, refers to substances that lackan affinity for water; tending to repel and not absorb water as well asnot dissolve in or mix with water.

The term “lipophilic”, as used herein, refers to compounds having anaffinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combininghydrophilic and lipophilic (hydrophobic) properties. “Amphiphilicmaterial” as used herein refers to a material containing a hydrophobicor more hydrophobic oligomer or polymer (e.g., biodegradable oligomer orpolymer) and a hydrophilic or more hydrophilic oligomer or polymer.

The term “targeting moiety”, as used herein, refers to a moiety thatbinds to or localizes to a specific locale. The moiety may be, forexample, a protein, nucleic acid, nucleic acid analog, carbohydrate, orsmall molecule. The locale may be a tissue, a particular cell type, or asubcellular compartment. In some embodiments, a targeting moiety canspecifically bind to a selected molecule.

The term “reactive coupling group”, as used herein, refers to anychemical functional group capable of reacting with a second functionalgroup to form a covalent bond. The selection of reactive coupling groupsis within the ability of those in the art. Examples of reactive couplinggroups can include primary amines (—NH₂) and amine-reactive linkinggroups such as isothiocyanates, isocyanates, acyl azides, NHS esters,sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates,aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenylesters. Most of these conjugate to amines by either acylation oralkylation. Examples of reactive coupling groups can include aldehydes(—COH) and aldehyde reactive linking groups such as hydrazides,alkoxyamines, and primary amines. Examples of reactive coupling groupscan include thiol groups (—SH) and sulfhydryl reactive groups such asmaleimides, haloacetyls, and pyridyl disulfides. Examples of reactivecoupling groups can include photoreactive coupling groups such as arylazides or diazirines. The coupling reaction may include the use of acatalyst, heat, pH buffers, light, or a combination thereof.

The term “protective group”, as used herein, refers to a functionalgroup that can be added to and/or substituted for another desiredfunctional group to protect the desired functional group from certainreaction conditions and selectively removed and/or replaced to deprotector expose the desired functional group. Protective groups are known tothe skilled artisan. Suitable protective groups may include thosedescribed in Greene and Wuts, Protective Groups in Organic Synthesis,(1991). Acid sensitive protective groups include dimethoxytrityl (DMT),tert-butylcarbamate (tBoc) and trifluoroacetyl (tFA). Base sensitiveprotective groups include 9-fluorenylmethoxycarbonyl (Fmoc), isobutyryl(iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other protective groupsinclude acetamidomethyl, acetyl, tert-amyloxycarbonyl, benzyl,benzyloxycarbonyl, 2-(4-biphenylyl)-2-propyloxycarbonyl,2-bromobenzyloxycarbonyl, tert-butyl₇ tert-butyloxycarbonyl,1-carbobenzoxamido-2,2.2-trifluoroethyl, 2,6-dichlorobenzyl,2-(3,5-dimethoxyphenyl)-2-propyl oxycarbonyl, 2,4-dinitrophenyl,dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl,4-methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl,α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl,benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester,p-nitrophenyl ester, phenyl ester, p-nitrocarbonate,p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester.

The term “activated ester”, as used herein, refers to alkyl esters ofcarboxylic acids where the alkyl is a good leaving group rendering thecarbonyl susceptible to nucleophilic attack by molecules bearing aminogroups. Activated esters are therefore susceptible to aminolysis andreact with amines to form amides. Activated esters contain a carboxylicacid ester group —CO₂R where R is the leaving group.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,C₃-C₃₀ for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms intheir ring structure, e.g., have 5, 6 or 7 carbons in the ringstructure. The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino,amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,aralkyl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, or from one to six carbon atoms in its backbonestructure. Likewise, “lower alkenyl” and “lower alkynyl” have similarchain lengths. In some embodiments, alkyl groups are lower alkyls. Insome embodiments, a substituent designated herein as alkyl is a loweralkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In some embodiments, the “alkylthio”moiety is represented by one of —S-alkyl, —S-alkenyl, and —S-alkynyl.Representative alkylthio groups include methylthio, and ethylthio. Theterm “alkylthio” also encompasses cycloalkyl groups, alkene andcycloalkene groups, and alkyne groups. “Arylthio” refers to aryl orheteroaryl groups. Alkylthio groups can be substituted as defined abovefor alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy, andtert-butoxy. An “ether” is two hydrocarbons covalently linked by anoxygen. Accordingly, the substituent of an alkyl that renders that alkylan ether is or resembles an alkoxyl, such as can be represented by oneof —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can be represented by—O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as definedbelow. The alkoxy and aroxy groups can be substituted as described abovefor alkyl.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈ or R₉ and R₁₀ taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R₉ or R₁₀ canbe a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not form animide. In still other embodiments, the term “amine” does not encompassamides, e.g., wherein one of R₉ and R₁₀ represents a carbonyl. Inadditional embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl or cycloalkyl, an alkenylor cycloalkenyl, or alkynyl. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted (asdescribed above for alkyl) or unsubstituted alkyl attached thereto,i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉ and R₁₀ are as defined above.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, for example, from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(Y)wherein Y is absent or is H, O, (C₁-C₁₀) alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic rings include, butare not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Heterocyclicgroups can optionally be substituted with one or more substituents atone or more positions as defined above for alkyl and aryl, for example,halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, and—CN.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, ancycloalkenyl, or an alkynyl, R′₁₁ represents a hydrogen, an alkyl, acycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl. Where X is anoxygen and R₁₁ or R′₁₁ is not hydrogen, the formula represents an“ester”. Where X is an oxygen and R₁₁ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₁₁ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen and R′₁₁ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′₁₁ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “monoester” as used herein refers to an analog of adicarboxylic acid wherein one of the carboxylic acids is functionalizedas an ester and the other carboxylic acid is a free carboxylic acid orsalt of a carboxylic acid. Examples of monoesters include, but are notlimited to, to monoesters of succinic acid, glutaric acid, adipic acid,suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Examples of heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium. Other useful heteroatomsinclude silicon and arsenic.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, for example, 1-14carbon atoms, and optionally include one or more heteroatoms such asoxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats. Representative substituents include alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy,phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio,substituted alkylthio, phenylthio, substituted phenylthio, arylthio,substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, peptide, and polypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e., a compound that does not spontaneously undergotransformation, for example, by rearrangement, cyclization, orelimination.

In a broad aspect, the permissible substituents include acyclic andcyclic, branched and unbranched, carbocyclic and heterocyclic, aromaticand nonaromatic substituents of organic compounds. Illustrativesubstituents include, for example, those described herein. Thepermissible substituents can be one or more and the same or differentfor appropriate organic compounds. The heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms.

In various embodiments, the substituent is selected from alkoxy,aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen,haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate,sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone,each of which optionally is substituted with one or more suitablesubstituents. In some embodiments, the substituent is selected fromalkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl,heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl,sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each ofthe alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl,arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl,haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide,sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can befurther substituted with one or more suitable substituents.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, thioketone, ester, heterocyclyl, —CN, aryl, aryloxy,perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl,heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters,carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl,alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl,carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl,alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl,perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, thesubstituent is selected from cyano, halogen, hydroxyl, and nitro.

The term “copolymer” as used herein, generally refers to a singlepolymeric material that is comprised of two or more different monomers.The copolymer can be of any form, for example, random, block, or graft.The copolymers can have any end-group, including capped or acid endgroups.

The term “mean particle size”, as used herein, generally refers to thestatistical mean particle size (diameter) of the particles in thecomposition. The diameter of an essentially spherical particle may bereferred to as the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer to the hydrodynamic diameter. As usedherein, the diameter of a non-spherical particle may refer to thelargest linear distance between two points on the surface of theparticle. Mean particle size can be measured using methods known in theart such as dynamic light scattering. Two populations can be said tohave a “substantially equivalent mean particle size” when thestatistical mean particle size of the first population of particles iswithin 20% of the statistical mean particle size of the secondpopulation of particles; for example, within 15%, or within 10%.

The terms “monodisperse” and “homogeneous size distribution”, as usedinterchangeably herein, describe a population of particles,microparticles, or nanoparticles all having the same or nearly the samesize. As used herein, a monodisperse distribution refers to particledistributions in which 90% of the distribution lies within 5% of themean particle size.

The terms “polypeptide,” “peptide” and “protein” generally refer to apolymer of amino acid residues. As used herein, the term also applies toamino acid polymers in which one or more amino acids are chemicalanalogs or modified derivatives of corresponding naturally-occurringamino acids or are unnatural amino acids. The term “protein”, asgenerally used herein, refers to a polymer of amino acids linked to eachother by peptide bonds to form a polypeptide for which the chain lengthis sufficient to produce tertiary and/or quaternary structure. The term“protein” excludes small peptides by definition, the small peptideslacking the requisite higher-order structure necessary to be considereda protein.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably to refer to a deoxyribonucleotide or ribonucleotidepolymer, in linear or circular conformation, and in either single- ordouble-stranded form. These terms are not to be construed as limitingwith respect to the length of a polymer. The terms can encompass knownanalogs of natural nucleotides, as well as nucleotides that are modifiedin the base, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general and unless otherwise specified, an analog of aparticular nucleotide has the same base-pairing specificity; i.e., ananalog of A will base-pair with T. The term “nucleic acid” is a term ofart that refers to a string of at least two base-sugar-phosphatemonomeric units. Nucleotides are the monomeric units of nucleic acidpolymers. The term includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA) in the form of a messenger RNA, antisense, plasmid DNA, partsof a plasmid DNA or genetic material derived from a virus. An antisensenucleic acid is a polynucleotide that interferes with the expression ofa DNA and/or RNA sequence. The term nucleic acids refers to a string ofat least two base-sugar-phosphate combinations. Natural nucleic acidshave a phosphate backbone. Artificial nucleic acids may contain othertypes of backbones, but contain the same bases as natural nucleic acids.The term also includes PNAs (peptide nucleic acids), phosphorothioates,and other variants of the phosphate backbone of native nucleic acids.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains atleast one function as the full-length protein, polypeptide or nucleicacid. A functional fragment can possess more, fewer, or the same numberof residues as the corresponding native molecule, and/or can contain oneor more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. The ability of a protein to interact with anotherprotein can be determined, for example, by co-immunoprecipitation,two-hybrid assays or complementation, e.g., genetic or biochemical. See,for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

As used herein, the term “linker” refers to a carbon chain that cancontain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. Linkersmay be substituted with various substituents including, but not limitedto, hydrogen atoms, alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, thioether, alkylthioether, thiol, and ureido groups. Thoseof skill in the art will recognize that each of these groups may in turnbe substituted. Examples of linkers include, but are not limited to,pH-sensitive linkers, protease cleavable peptide linkers, nucleasesensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers,photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers(e.g., esterase cleavable linker), ultrasound-sensitive linkers, andx-ray cleavable linkers.

The term “pharmaceutically acceptable counter ion” refers to apharmaceutically acceptable anion or cation. In various embodiments, thepharmaceutically acceptable counter ion is a pharmaceutically acceptableion. For example, the pharmaceutically acceptable counter ion isselected from citrate, malate, acetate, oxalate, chloride, bromide,iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate; ethanesulfonate, benzenesulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)). In some embodiments, thepharmaceutically acceptable counter ion is selected from chloride,bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,citrate, malate, acetate, oxalate, acetate, and lactate. In particularembodiments, the pharmaceutically acceptable counter ion is selectedfrom chloride, bromide, iodide, nitrate, sulfate, bisulfate, andphosphate.

The term “pharmaceutically acceptable salt(s)” refers to salts of acidicor basic groups that may be present in compounds used in the presentcompositions. Compounds included in the present compositions that arebasic in nature are capable of forming a variety of salts with variousinorganic and organic acids. The acids that may be used to preparepharmaceutically acceptable acid addition salts of such basic compoundsare those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to sulfate, citrate, malate, acetate, oxalate, chloride,bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate,tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds includedin the present compositions that include an amino moiety may formpharmaceutically acceptable salts with various amino acids, in additionto the acids mentioned above. Compounds included in the presentcompositions, that are acidic in nature are capable of forming basesalts with various pharmacologically acceptable cations. Examples ofsuch salts include alkali metal or alkaline earth metal salts and,particularly, calcium, magnesium, sodium, lithium, zinc, potassium, andiron salts.

If the compounds described herein are obtained as an acid addition salt,the free base can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt,particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare non-toxic pharmaceutically acceptable additionsalts.

A pharmaceutically acceptable salt can be derived from an acid selectedfrom 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid,2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoicacid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid,camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid(hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamicacid, citric acid, cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaricacid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid,glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid,isethionic, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonicacid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmiticacid, pamoic acid, pantothenic, phosphoric acid, proprionic acid,pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinicacid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonicacid, trifluoroacetic, and undecylenic acid.

The term “bioavailable” is art-recognized and refers to a form of thesubject invention that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

II. Conjugates

Conjugates include an active agent or prodrug thereof attached to atargeting moiety, e.g., a molecule that can bind to an SSTR, by alinker. The conjugates can be a conjugate between a single active agentand a single targeting moiety, e.g., a conjugate having the structureX—Y—Z where X is the targeting moiety, Y is the linker, and Z is theactive agent.

In some embodiments the conjugate contains more than one targetingmoiety, more than one linker, more than one active agent, or anycombination thereof. The conjugate can have any number of targetingmoieties, linkers, and active agents. The conjugate can have thestructure X—Y—Z—Y—X, (X-Y)_(n)—Z, X—(Y—Z)_(n), X—Y—Z_(n), (X—Y—Z)_(n),(X—Y—Z—Y)_(n)—Z where X is a targeting moiety, Y is a linker, Z is anactive agent, and n is an integer between 1 and 50, between 2 and 20,for example, between 1 and 5. Each occurrence of X, Y, and Z can be thesame or different, e.g., the conjugate can contain more than one type oftargeting moiety, more than one type of linker, and/or more than onetype of active agent.

The conjugate can contain more than one targeting moiety attached to asingle active agent. For example, the conjugate can include an activeagent with multiple targeting moieties each attached via a differentlinker. The conjugate can have the structure X—Y—Z—Y—X where each X is atargeting moiety that may be the same or different, each Y is a linkerthat may be the same or different, and Z is the active agent.

The conjugate can contain more than one active agent attached to asingle targeting moiety. For example the conjugate can include atargeting moiety with multiple active agents each attached via adifferent linker. The conjugate can have the structure Z—Y—X-Y—Z where Xis the targeting moiety, each Y is a linker that may be the same ordifferent, and each Z is an active agent that may be the same ordifferent.

A. Active Agents

A conjugate as described herein contains at least one active agent (afirst active agent). The conjugate can contain more than one activeagent, that can be the same or different from the first active agent.The active agent can be a therapeutic, prophylactic, diagnostic, ornutritional agent. A variety of active agents are known in the art andmay be used in the conjugates described herein. The active agent can bea protein or peptide, small molecule, nucleic acid or nucleic acidmolecule, lipid, sugar, glycolipid, glycoprotein, lipoprotein, orcombination thereof. In some embodiments, the active agent is anantigen, an adjuvant, radioactive, an imaging agent (e.g., a fluorescentmoiety) or a polynucleotide. In some embodiments the active agent is anorganometallic compound.

Anti-Cancer-Agents

The active agent can be a cancer therapeutic. Cancer therapeuticsinclude, for example, death receptor agonists such as the TNF-relatedapoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand orantibody that binds or activates a death receptor or otherwise inducesapoptosis. Suitable death receptors include, but are not limited to,TNFR1, Fas, DR3, DR4, DR5, DR6, LTβR and combinations thereof.

Cancer therapeutics such as chemotherapeutic agents, cytokines,chemokines, and radiation therapy agents can be used as active agents.Chemotherapeutic agents include, for example, alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumor agents. Such agents typically affectcell division or DNA synthesis and function. Additional examples oftherapeutics that can be used as active agents include monoclonalantibodies and the tyrosine kinase inhibitors e.g. imatinib mesylate,which directly targets a molecular abnormality in certain types ofcancer (e.g., chronic myelogenous leukemia, gastrointestinal stromaltumors).

Chemotherapeutic agents include, but are not limited to cisplatin,carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxoland derivatives thereof, irinotecan, topotecan, amsacrine, etoposide,etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab,cetuximab, and rituximab, bevacizumab, and combinations thereof. Any ofthese may be used as an active agent in a conjugate.

In some embodiments, the active agent can be 20-epi-1,25dihydroxyvitamin D3, 4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol,abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine,acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists,altretamine, ambamustine, ambomycin, ametantrone acetate, amidox,amifostine, aminoglutethimide, aminolevulinic acid, amrubicin,amsacrine, anagrelide, anastrozole, andrographolide, angiogenesisinhibitors, antagonist D, antagonist G, antarelix, anthramycin,anti-dorsalizing morphogenetic protein-1, anti estrogen, antineoplaston,antisense oligonucleotides, aphidicolin glycinate, apoptosis genemodulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA,arginine deaminase, asparaginase, asperlin, asulacrine, atamestane,atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine,azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin IIIderivatives, balanol, batimastat, benzochlorins, benzodepa,benzoylstaurosporine, beta lactam derivatives, beta-alethine,betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide,bisantrene, bisantrene hydrochloride, bisaziridinylspermine, bisnafide,bisnafide dimesylate, bistratene A, bizelesin, bleomycin, bleomycinsulfate, BRC/ABL antagonists, breflate, brequinar sodium, bropirimine,budotitane, busulfan, buthionine sulfoximine, cabazitaxel, cactinomycin,calcipotriol, calphostin C, calusterone, camptothecin, camptothecinderivatives, canarypox IL-2, capecitabine, caracemide, carbetimer,carboplatin, carboxamide-amino-tri azole, carboxyamidotriazole, carestM3, carmustine, earn 700, cartilage derived inhibitor, carubicinhydrochloride, carzelesin, casein kinase inhibitors, castano spermine,cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins,chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cisplatin,cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycinA, collismycin B, combretastatin A4, combretastatin analog, conagenin,crambescidin 816, crisnatol, crisnatol mesylate, cryptophycin 8,cryptophycin A derivatives, curacin A, cyclopentanthraquinones,cyclophosphamide, cycloplatam, cypemycin, cytarabine, cytarabineocfosfate, cytolytic factor, cytostatin, dacarbazine, dacliximab,dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B,deslorelin, dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenylspiromustine, docetaxel, docosanol, dolasetron, doxifluridine,doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifenecitrate, dromostanolone propionate, dronabinol, duazomycin, duocarmycinSA, ebselen, ecomustine, edatrexate, edelfosine, edrecolomab,eflornithine, eflornithine hydrochloride, elemene, elsamitrucin,emitefur, enloplatin, enpromate, epipropidine, epirubicin, epirubicinhydrochloride, epristeride, erbulozole, erythrocyte gene therapy vectorsystem, esorubicin hydrochloride, estramustine, estramustine analog,estramustine phosphate sodium, estrogen agonists, estrogen antagonists,etanidazole, etoposide, etoposide phosphate, etoprine, exemestane,fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim,finasteride, flavopiridol, flezelastine, floxuridine, fluasterone,fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride,fluorouracil, flurocitabine, forfenimex, formestane, fosquidone,fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat,imidazoacridones, imiquimod, immunostimulant peptides, insulin-likegrowth factor-1 receptor inhibitor, interferon agonists, interferonalpha-2A, interferon alpha-2B, interferon alpha-N1, interferon alpha-N3,interferon beta-IA, interferon gamma-IB, interferons, interleukins,iobenguane, iododoxorubicin, iproplatin, irinotecan, irinotecanhydrochloride, iroplact, irsogladine, isobengazole, isohomohalicondrinB, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate,lanreotide, larotaxel, lanreotide acetate, leinamycin, lenograstim,lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor,leukocyte alpha interferon, leuprolide acetate,leuprolide/estrogen/progesterone, leuprorelin, levamisole, liarozole,liarozole hydrochloride, linear polyamine analog, lipophilicdisaccharide peptide, lipophilic platinum compounds, lissoclinamide 7,lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine,lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin,loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lyticpeptides, maitansine, mannostatin A, marimastat, masoprocol, maspin,matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine,maytansinoid, mertansine (DM1), mechlorethamine hydrochloride, megestrolacetate, melengestrol acetate, melphalan, menogaril, merbarone,mercaptopurine, meterelin, methioninase, methotrexate, methotrexatesodium, metoclopramide, metoprine, meturedepa, microalgal protein kinaseC inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim,mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin,mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycinanalogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid a/mycobacterium cell wall SK, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, mycophenolic acid, myriaporone, n-acetyldinaline,nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, nocodazole, nogalamycin, n-substitutedbenzamides, 06-benzylguanine, octreotide, okicenone, oligonucleotides,onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxelanalogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin,pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine,pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfatesodium, pentostatin, pentrozole, peplomycin sulfate, perflubron,perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,phosphatase inhibitors, picibanil, pilocarpine hydrochloride,pipobroman, piposulfan, pirarubicin, piritrexim, piroxantronehydrochloride, placetin A, placetin B, plasminogen activator inhibitor,platinum(IV) complexes, platinum compounds, platinum-triamine complex,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2,prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,puromycin, puromycin hydrochloride, purpurins, pyrazofurin,pyrazoloacridine, pyridoxylated hemoglobin polyoxy ethylene conjugate,RAF antagonists, raltitrexed, ramosetron, RAS farnesyl proteintransferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptinedemethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes,RII retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex,rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin,sarcnu, sarcophytol A, sargramostim, SDI 1 mimetics, semustine,senescence derived inhibitor 1, sense oligonucleotides, siRNA, signaltransduction inhibitors, signal transduction modulators, simtrazene,single chain antigen binding protein, sizofiran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin,spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, streptonigrin, streptozocin,stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifenmethiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride,temoporfin, temozolomide, teniposide, teroxirone, testolactone,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyletiopurpurin, tirapazamine, titanocene dichloride, topotecanhydrochloride, topsentin, toremifene, toremifene citrate, totipotentstem cell factor, translation inhibitors, trestolone acetate, tretinoin,triacetyluridine, triciribine, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tropisetron, tubulozolehydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBCinhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derivedgrowth inhibitory factor, urokinase receptor antagonists, vapreotide,variolin B, velaresol, veramine, verdins, verteporfin, vinblastinesulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidinesulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine,vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidinesulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.

In some embodiments the active agent is cabazitaxel, or an analog,derivative, prodrug, or pharmaceutically acceptable salt thereof.

The active agent can be an inorganic or organometallic compoundcontaining one or more metal centers. In some examples, the compoundcontains one metal center. The active agent can be, for example, aplatinum compound, a ruthenium compound (e.g., trans-[RuCl₂ (DMSO)₄], ortrans-[RuCl₄(imidazole)₂, etc.), cobalt compound, copper compound, oriron compounds.

In certain embodiments, the active agent of the conjugate comprises apredetermined molar weight percentage from about 1% to about 10%, orabout 10% to about 20%, or about 20% to about 30%, or about 30% to about40%, or about 40% to about 50%, or about 50% to about 60%, or about 60%to about 70%, or about 70% to about 80%, or about 80% to about 90%, orabout 90% to about 99% such that the sum of the molar weight percentagesof the components of the conjugate is 100%. The amount of activeagent(s) of the conjugate may also be expressed in terms of proportionto the targeting ligand(s). For example, the present teachings provide aratio of active agent to ligand of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

B. Targeting Moieties

Targeting ligands (also referred to as targeting moieties) as describedherein include any molecule that can bind one or more SSTRs, e.g., humanSSTR1, SSTR2, SSTR3, SSTR4, or SSTR5. Such targeting ligands can bepeptides, antibody mimetics, nucleic acids (e.g., aptamers),polypeptides (e.g., antibodies), glycoproteins, small molecules,carbohydrates, or lipids. In some embodiments, the targeting moiety issomatostatin or a somatostatin analog.

The cytotoxic or therapeutic conjugates of the invention can employ anysomatostatin analog that binds somatostatin receptor. In someembodiments, the somatostatin analog portion of the conjugate containsbetween 8 and 18 amino acids, and includes the core sequence:cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys] (SEQ ID NO:1) orcyclo[Cys-Tyr-D-Trp-Lys-Thr-Cys] (SEQ ID NO. 2). For example, theC-terminus of the analog is Thr-NH2.

In some embodiments, the targeting moiety, X, may be selected fromsomatostatin, octreotide, Tyr³-octreotate (TATE), vapreotide,cyclo(AA-Tyr-DTrp-Lys-Thr-Phe) where AA is α-N-Me lysine or N-Meglutamic acid, pasireotide, lanreotide, seglitide, or any other exampleof somatostatin receptor binding ligands. In some embodiments, thetargeting moiety is a somatostatin receptor binding moiety that binds tosomatostatin receptors 2 and/or 5. In some embodiments, X binds to thelinker moiety Y at the C-terminal. In some embodiments, X binds to thelinker moiety Y at the N-terminal. In some embodiments, the targetingmoiety X comprises at least one D-Phe residue and the phenyl ring of theD-Phe residue of the targeting moiety X has been replaced by alinker-containing moiety.

Examples of somatostatin analogs that are peptides useful in the presentinvention are described herein. Further examples useful somatostatinanalogs are disclosed in publications set forth below, each of which ishereby incorporated by reference in its entirety:

-   PCT Application No. WO 03/057214 (2003)-   U.S. Application No. 20030191134 (2003)-   U.S. Application No. 20030083241 (2003)-   U.S. Pat. No. 6,316,414 (2001)-   PCT Application No. WO 02/10215 (2002)-   PCT Application No. WO 99/22735 (1999)-   PCT Application No. WO 98/08100 (1998)-   PCT Application No. WO 98/44921 (1998)-   PCT Application No. WO 98/45285 (1998)-   PCT Application No. WO 98/44922 (1998)-   EP Application No. P5164 EU (Inventor: G. Keri);-   Van Binst, G. et al., Peptide Research, 1992, 5:8;-   Horvath, A. et al., Abstract, “Conformations of Somatostatin Analogs    Having Antitumor Activity”, 22nd European peptide Symposium, Sep.    13-19, 1992, Interlaken, Switzerland;-   PCT Application No. WO 91/09056 (1991);-   EP Application No. 0 363 589 A2 (1990);-   U.S. Pat. No. 4,904,642 (1990);-   U.S. Pat. No. 4,871,717 (1989);-   U.S. Pat. No. 4,853,371 (1989);-   U.S. Pat. No. 4,725,577 (1988);-   U.S. Pat. No. 4,684,620 (1987);-   U.S. Pat. No. 4,650,787 (1987);-   U.S. Pat. No. 4,603,120 (1986);-   U.S. Pat. No. 4,585,755 (1986);-   EP Application No. 0 203 031 A2 (1986);-   U.S. Pat. No. 4,522,813 (1985);-   U.S. Pat. No. 4,486,415 (1984);-   U.S. Pat. No. 4,485,101 (1984);-   U.S. Pat. No. 4,435,385 (1984);-   U.S. Pat. No. 4,395,403 (1983);-   U.S. Pat. No. 4,369,179 (1983);-   U.S. Pat. No. 4,360,516 (1982);-   U.S. Pat. No. 4,358,439 (1982);-   U.S. Pat. No. 4,328,214 (1982);-   U.S. Pat. No. 4,316,890 (1982);-   U.S. Pat. No. 4,310,518 (1982);-   U.S. Pat. No. 4,291,022 (1981);-   U.S. Pat. No. 4,238,481 (1980);-   U.S. Pat. No. 4,235,886 (1980);-   U.S. Pat. No. 4,224,199 (1980);-   U.S. Pat. No. 4,211,693 (1980);-   U.S. Pat. No. 4,190,648 (1980);-   U.S. Pat. No. 4,146,612 (1979);-   U.S. Pat. No. 4,133,782 (1979);-   U.S. Pat. No. 5,506,339 (1996);-   U.S. Pat. No. 4,261,885 (1981);-   U.S. Pat. No. 4,728,638 (1988);-   U.S. Pat. No. 4,282,143 (1981);-   U.S. Pat. No. 4,215,039 (1980);-   U.S. Pat. No. 4,209,426 (1980);-   U.S. Pat. No. 4,190,575 (1980);-   EP Patent No. 0 389 180 (1990);-   EP Application No. 0 505 680 (1982);-   EP Application No. 0 083 305 (1982);-   EP Application No. 0 030 920 (1980);-   PCT Application No. WO 88/05052 (1988);-   PCT Application No. WO 90/12811 (1990);-   PCT Application No. WO 97/01579 (1997);-   PCT Application No. WO 91/18016 (1991);-   U.K. Application No. GB 2,095,261 (1981);-   French Application No. FR 2,522,655 (1983); and-   PCT Application No. WO 04/093807 (2004).-   U.S. Pat. No. 5,620,955 (1997)-   U.S. Pat. No. 5,723,578 (1998)-   U.S. Pat. No. 5,843,903 (1998)-   U.S. Pat. No. 5,877,277 (1999)-   U.S. Pat. No. 6,156,725 (2000)-   U.S. Pat. No. 6,307,017 (2001)-   PCT Application No. WO 90/03980 (1990)-   PCT Application No. WO 91/06563 (1991)-   PCT Application No. WO 91/17181 (1991)-   PCT Application No. WO 94/02018 (1994)-   PCT Application No. WO 94/21674 (1994)-   PCT Application No. WO 04/093807 (2004);

Methods for synthesizing somatostatin peptides and analogs are welldocumented and are within the ability of a person of ordinary skill inthe art as exemplified in the references listed supra. Further syntheticprocedures are provided in the following examples. The followingexamples also illustrate methods for synthesizing the targeted cytotoxiccompounds of the present invention. Specific targeting of therapeutic orcytotoxic agents allows selective destruction of a tumor expressing areceptor specific for a biologically active peptide. For example, atumor expressing a somatostatin receptor includes a neoplasm of thelung, breast, prostate, colon, brain, gastrointestinal tract,neuroendocrine axis, liver, or kidney (see Schaer et al., Int. J.Cancer, 70:530-537, 1997; Chave et al., Br. J. Cancer 82(1):124-130,2000; Evans et al., Br. J. Cancer 75(6):798-803, 1997).

In some embodiments, the targeting moiety has therapeutic features,e.g., the targeting moiety is cytotoxic or anti-angiogenic. In someembodiments, a targeting moiety has some increased affinity for tumorvasculature, or angiogenic blood vessels, e.g., those that over-expresssomatostatin receptors (see Denzler and Reubi, Cancer 85:188-198, 1999;Gulec et al., J. Surg. Res. 97(2):131-137, 2001; Woltering et al., J.Surg. Res. 50:245, 1991).

In some embodiments, the targeting moiety, e.g., somatostatin analog,used in the invention is hydrophilic, and is therefore water soluble. Insome embodiments, such conjugates and particles containing suchconjugates are used in treatment paradigms in which this feature isuseful, e.g., compared to conjugates comprising hydrophobic analogs.Hydrophilic analogs described herein can be soluble in blood,cerebrospinal fluid, and other bodily fluids, as well as in urine, whichmay facilitate excretion by the kidneys. This feature can be useful,e.g., in the case of a composition that would otherwise exhibitundesirable liver toxicity. The invention also discloses specifichydrophilic elements (e.g., incorporation of a PEG linker, and otherexamples in the art) for incorporation into peptide analogs, allowingmodulation of the analog's hydrophilicity to adjust for the chemical andstructural nature of the various conjugated cytotoxic agents, e.g.,conjugate 6 infra.

In some embodiments, the targeting moiety is an antibody mimetic such asa monobody, e.g., an ADNECTIN™ (Bristol-Myers Squibb, New York, N.Y.),an Affibody® (Affibody AB, Stockholm, Sweden), Affilin, nanofitin(affitin, such as those described in WO 2012/085861, an Anticalin™, anavimers (avidity multimers), a DARPin™, a Fynomer™, Centyrin™ and aKunitz domain peptide. In certain cases, such mimetics are artificialpeptides or proteins with a molar mass of about 3 to 20 kDa. Nucleicacids and small molecules may be antibody mimetic.

In another example, a targeting moiety can be an aptamer, which isgenerally an oligonucleotide (e.g., DNA, RNA, or an analog or derivativethereof) that binds to a particular target, such as a polypeptide. Insome embodiments, the targeting moiety is a polypeptide (e.g., anantibody that can specifically bind a tumor marker). In certainembodiments, the targeting moiety is an antibody or a fragment thereof.In certain embodiments, the targeting moiety is an Fc fragment of anantibody.

In certain embodiments, the targeting moiety or moieties of theconjugate are present at a predetermined molar weight percentage fromabout 0.1% to about 10%, or about 1% to about 10%, or about 10% to about20%, or about 20% to about 30%, or about 30% to about 40%, or about 40%to about 50%, or about 50% to about 60%, or about 60% to about 70%, orabout 70% to about 80%, or about 80% to about 90%, or about 90% to about99% such that the sum of the molar weight percentages of the componentsof the conjugate is 100%. The amount of targeting moieties of theconjugate may also be expressed in terms of proportion to the activeagent(s), for example, in a ratio of ligand to active agent of about10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5,1:6, 1:7, 1:8, 1:9, or 1:10.

C. Linkers

The conjugates contain one or more linkers attaching the active agentsand targeting moieties. The linker, Y, is bound to one or more activeagents and one or more targeting ligands to form a conjugate. The linkerY is attached to the targeting moiety X and the active agent Z byfunctional groups independently selected from an ester bond, disulfide,amide, acylhydrazone, ether, carbamate, carbonate, and urea.Alternatively the linker can be attached to either the targeting ligandor the active drug by a non-cleavable group such as provided by theconjugation between a thiol and a maleimide, an azide and an alkyne. Thelinker is independently selected from the group consisting alkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of thealkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groupsoptionally is substituted with one or more groups, each independentlyselected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl,ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl,alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, whereineach of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide,carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,heteroaryl, or heterocyclyl is optionally substituted with one or moregroups, each independently selected from halogen, cyano, nitro,hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide,carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,heteroaryl, heterocyclyl.

In some embodiments, the linker comprises a cleavable functionality thatis cleavable. The cleavable functionality may be hydrolyzed in vivo ormay be designed to be hydrolyzed enzymatically, for example by CathepsinB. A “cleavable” linker, as used herein, refers to any linker which canbe cleaved physically or chemically. Examples for physical cleavage maybe cleavage by light, radioactive emission or heat, while examples forchemical cleavage include cleavage by re-dox-reactions, hydrolysis,pH-dependent cleavage or cleavage by enzymes.

In some embodiments the alkyl chain of the linker may optionally beinterrupted by one or more atoms or groups selected from —O—, —C(═O)—,—NR, —O—C(═O)—NR—, —S—, —S—S—. The linker may be selected fromdicarboxylate derivatives of succinic acid, glutaric acid or diglycolicacid. In some embodiments, the linker Y may be X′—R¹—Y′—R²—Z′ and theconjugate can be a compound according to Formula Ia:

wherein X is a targeting moiety defined above; Z is an active agent; X′,R¹, Y′, R² and Z′ are as defined herein.

X′ is either absent or independently selected from carbonyl, amide,urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or morenatural or unnatural amino acids, thio or succinimido; R¹ and R² areeither absent or comprised of alkyl, substituted alkyl, aryl,substituted aryl, polyethylene glycol (2-30 units); Y′ is absent,substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol(2-30 units) or an amide; Z′ is either absent or independently selectedfrom carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy,arylamino, thio or succinimido. In some embodiments, the linker canallow one active agent molecule to be linked to two or more ligands, orone ligand to be linked to two or more active agent molecules.

In some embodiments, the linker Y may be A_(m) and the conjugate can bea compound according to Formula Ib:

wherein A is defined herein, m=0-20.

A in Formula Ia is a spacer unit, either absent or independentlyselected from the following substituents. For each substituent, thedashed lines represent substitution sites with X, Z or anotherindependently selected unit of A wherein the X, Z, or A can be attachedon either side of the substituent:

wherein z=0-40, R is H or an optionally substituted alkyl group, and R′is any side chain found in either natural or unnatural amino acids.

In some embodiments, the conjugate may be a compound according toFormula Ic:

wherein A is defined above, m=0-40, n=0-40, x=1-5, y=1-5, and C is abranching element defined herein.

C in Formula Ic is a branched unit containing three to sixfunctionalities for covalently attaching spacer units, ligands, oractive drugs, selected from amines, carboxylic acids, thiols, orsuccinimides, including amino acids such as lysine, 2,3-diaminopropanoicacid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, andcysteine.

Non-limiting examples of conjugates of the present invention include thefollowing compounds:

In some embodiments, the active agent Z is DM1 and the somatostatinreceptor binding agent X is selected from somatostatin,cyclo(AA-Tyr-DTrp-Lys-Thr-Phe), vapreotide or TATE. In some embodiments,DM1 is connected to the C-terminus of X with the linker Y. In someembodiments, DM1 is connected to the N-terminus of X with the linker Y.In some embodiments, DM1 is connected to X with the linker Y, whereinthe targeting moiety X comprises at least one D-Phe residue and thephenyl ring of the D-Phe residue has been replaced by a group containinglinker Y.

Non-limiting examples of conjugates comprising DM1, referred to as DM1conjugates of the invention, include the following compounds:

1) Cyclo(AA-Tyr-DTrp-Lys-Thr-Phe)-Based DM1 Conjugates

In some embodiments, cyclo(AA-Tyr-DTrp-Lys-Thr-Phe) is used as asomatostatin receptor targeting moiety and the conjugates have a generalstructure of:

In some embodiments, the targeting moiety contains an amino acid capableof making an amide bond. In some embodiments, the linker is bound to thetargeting moiety via an amide bond, i.e., —NH—CO—, or —CO—NH— (thehydrogen on the nitrogen may be substituted). In some embodiments, thelinker is not bound to the targeting moiety via an amide bond. In someembodiments, the linker includes an amide bond, i.e., —NH—CO—, or—CO—NH— (the hydrogen on the nitrogen may be substituted).

Non-limiting examples of conjugates comprising cyclo(AA-Tyr-DTrp-LysThr-Phe) and DM1 are shown in Table 1:

TABLE 1 cyclo(AA-Tyr-DTrp-Lys-Thr-Phe)-based Conjugates Linker* Fullstructure

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32 *In order to show linker structures, the somatostatin receptortargeting moiety is referredto as ligand in the structures.2) C-Terminal DM1 Conjugates:

In some embodiments, the somatostatin receptor targeting moiety is apeptide and the linker binds to the C-terminus of the somatostatinreceptor targeting moiety. In some embodiments, the somatostatinreceptor targeting moiety is TATE or a TATE derivative, wherein thelinker binds to the C-terminus of TATE or the TATE derivative. TheC-terminal DM1 conjugates have a general structure of:

wherein R is selected from H, alkyl, aryl, carbonyl, amide, alcohol, oramine, optionally substituted with one or more groups; andAr₁ and Ar₂ are independently selected from heterocyclyl, aryl, andheteroaryl groups optionally substituted with one or more groups.

In some embodiments, the covalent bond connecting the linker and theC-terminus of the somatostatin receptor targeting moiety is an amidebond.

Non-limiting examples of DM1 conjugates wherein the linker binds to theC-terminus of the somatostatin receptor targeting moiety, wherein thesomatostatin receptor targeting moiety is TATE, are shown in Table 2:

TABLE 2 C-terminal DM1-TATE conjugates R Ar1 Ar2 Linker* Full StructureH

H

H

H

H

H

H

H

H

H

H

H

H

H

*In order to show linker structures, the somatostatin receptor targetingmoiety is referred to as ligand in the structures.3) N-Terminal DM1 Conjugates

In some embodiments, the somatostatin receptor targeting moiety is apeptide and the linker binds to the N-terminus of the somatostatinreceptor targeting moiety. In some embodiments, the target moiety isselected from octreotide, vapreotide, and TATE. In some embodiments, thecovalent bond connecting the linker and the N-terminal of thesomatostatin receptor targeting moiety is an amide bond, i.e., —NH—CO—.In some embodiments, the linker binds to the N-terminus of thesomatostatin receptor targeting moiety via an amine bond, i.e.,—NH—CH2-(hydrogen on the carbon may be substituted). In someembodiments, the linker binds to the N-terminus of the somatostatinreceptor targeting moiety via a urea bond, i.e. —NH—CO—NH—. TheN-terminal DM1 conjugate has a general structure of:

wherein R₁ and R₂ are independently selected from H, OH, alkyl, aryl,carbonyl, ester, amide, ether, alcohol, or amine, optionally substitutedwith one or more groups; and Ar₁ is selected from heterocyclyl, aryl,and heteroaryl groups optionally substituted with one or more groups. Insome embodiments, at least one of R1 or R2 comprises DM1.

Non-limiting examples of DM1 conjugates wherein the linker binds to theN-terminus of the somatostatin receptor targeting moiety are shown inTable 3:

TABLE 3 N-terminal DM1 conjugates R₁ R₂ Ar₁ Linker* Full structure

—OH

—Me

—OH

—OH

—OH

—Me

—OH

—OH

—OH

—OH

—OH

—OH

—OH

—OH

—OH

—OH

—OH

—Me

—OH

—OH

—OH

*In order to show linker structures, the somatostatin receptor targetingmoiety is referred to as ligand in the structures.4) D-Phe Replacement DM1 Conjugates

In some embodiments, the somatostatin receptor targeting moiety is atargeting ligand such as octreotide or TATE, wherein the phenyl ring ofthe D-Phe residue of the targeting ligand has been replaced by alinker-containing moiety. The D-Phe replacement DM1 conjugate has ageneral structure of:

Wherein R is selected from H, OH, alkyl, aryl, carbonyl, ester, amide,ether, alcohol, or amine, optionally substituted with one or moregroups. In some embodiments, R comprises DM1.

Non-limiting examples of DM1 conjugates wherein the phenyl ring of theD-Phe residue of the targeting ligand has been replaced by alinker-containing moiety are shown in Table 4:

TABLE 4 D-Phe replacement conjugates R Linker* Full structure H

H

H

H

*In order to show linker structures, the somatostatin receptor targetingmoiety is referred to as ligand in the structures.

III. Particles

Particles containing one or more conjugates can be polymeric particles,lipid particles, solid lipid particles, inorganic particles, orcombinations thereof (e.g., lipid stabilized polymeric particles). Insome embodiments, the particles are polymeric particles or contain apolymeric matrix. The particles can contain any of the polymersdescribed herein or derivatives or copolymers thereof. The particlesgenerally contain one or more biocompatible polymers. The polymers canbe biodegradable polymers. The polymers can be hydrophobic polymers,hydrophilic polymers, or amphiphilic polymers. In some embodiments, theparticles contain one or more polymers having an additional targetingmoiety attached thereto.

The size of the particles can be adjusted for the intended application.The particles can be nanoparticles or microparticles. The particle canhave a diameter of about 10 nm to about 10 microns, about 10 nm to about1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, orabout 25 nm to about 250 nm. In some embodiments the particle is ananoparticle having a diameter from about 25 nm to about 250 nm. It isunderstood by those in the art that a plurality of particles will have arange of sizes and the diameter is understood to be the median diameterof the particle size distribution.

In various embodiments, a particle may be a nanoparticle, i.e., theparticle has a characteristic dimension of less than about 1 micrometer,where the characteristic dimension of a particle is the diameter of aperfect sphere having the same volume as the particle. The plurality ofparticles can be characterized by an average diameter (e.g., the averagediameter for the plurality of particles). In some embodiments, thediameter of the particles may have a Gaussian-type distribution. In someembodiments, the plurality of particles have an average diameter of lessthan about 300 nm, less than about 250 nm, less than about 200 nm, lessthan about 150 nm, less than about 100 nm, less than about 50 nm, lessthan about 30 nm, less than about 10 nm, less than about 3 nm, or lessthan about 1 nm. In some embodiments, the particles have an averagediameter of at least about 5 nm, at least about 10 nm, at least about 30nm, at least about 50 nm, at least about 100 nm, at least about 150 nm,or greater. In certain embodiments, the plurality of the particles hasan average diameter of about 10 nm, about 25 nm, about 50 nm, about 100nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500nm, or the like. In some embodiments, the plurality of particles has anaverage diameter between about 10 nm and about 500 nm, between about 50nm and about 400 nm, between about 100 nm and about 300 nm, betweenabout 150 nm and about 250 nm, between about 175 nm and about 225 nm, orthe like. In some embodiments, the plurality of particles has an averagediameter between about 10 nm and about 500 nm, between about 20 nm andabout 400 nm, between about 30 nm and about 300 nm, between about 40 nmand about 200 nm, between about 50 nm and about 175 nm, between about 60nm and about 150 nm, between about 70 nm and about 130 nm, or the like.For example, the average diameter can be between about 70 nm and 130 nm.In some embodiments, the plurality of particles has an average diameterbetween about 20 nm and about 220 nm, between about 30 nm and about 200nm, between about 40 nm and about 180 nm, between about 50 nm and about170 nm, between about 60 nm and about 150 nm, or between about 70 nm andabout 130 nm. In one embodiment, the particles have a size of 40 to 120nm with a zeta potential close to 0 mV at low to zero ionic strengths (1to 10 mM), with zeta potential values between +5 to −5 mV, and azero/neutral or a small −ve surface charge.

A. Conjugates

The particles contain one or more conjugates as described above. Theconjugates can be present on the interior of the particle, on theexterior of the particle, or both. The particles may comprisehydrophobic ion-pairing complexes or hydrophobic ion-pairs formed by oneor more conjugates described above and counterions.

Hydrophobic ion-pairing (HIP) is the interaction between a pair ofoppositely charged ions held together by Coulombic attraction. HIP, asused here in, refers to the interaction between the conjugate of thepresent invention and its counterions, wherein the counterion is not H⁺or HO⁻ ions. Hydrophobic ion-pairing complex or hydrophobic ion-pair, asused herein, refers to the complex formed by the conjugate of thepresent invention and its counterions. In some embodiments, thecounterions are hydrophobic. In some embodiments, the counterions areprovided by a hydrophobic acid or a salt of a hydrophobic acid. In someembodiments, the counterions are provided by bile acids or salts, fattyacids or salts, lipids, or amino acids. In some embodiments, thecounterions are negatively charged (anionic). Non-limited examples ofnegative charged counterions include the counterions sodiumsulfosuccinate (AOT), sodium oleate, sodium dodecyl sulfate (SDS), humanserum albumin (HSA), dextran sulphate, sodium deoxycholate, sodiumcholate, anionic lipids, amino acids, or any combination thereof.Without wishing to be bound by any theory, in some embodiments, HIP mayincrease the hydrophobicity and/or lipophilicity of the conjugate of thepresent invention. In some embodiments, increasing the hydrophobicityand/or lipophilicity of the conjugate of the present invention may bebeneficial for particle formulations and may provide higher solubilityof the conjugate of the present invention in organic solvents. Withoutwishing to be bound by any theory, it is believed that particleformulations that include HIP pairs have improved formulationproperties, such as drug loading and/or release profile. Without wishingto be bound by any theory, in some embodiments, slow release of theconjugate of the invention from the particles may occur, due to adecrease in the conjugate's solubility in aqueous solution. In addition,without wishing to be bound by any theory, complexing the conjugate withlarge hydrophobic counterions may slow diffusion of the conjugate withina polymeric matrix. In some embodiments, HIP occurs without covalentconfutation of the counterion to the conjugate of the present invention.

Without wishing to be bound by any theory, the strength of HIP mayimpact the drug load and release rate of the particles of the invention.In some embodiments, the strength of the HIP may be increased byincreasing the magnitude of the difference between the pKa of theconjugate of the present invention and the pKa of the agent providingthe counterion. Also without wishing to be bound by any theory, theconditions for ion pair formation may impact the drug load and releaserate of the particles of the invention.

In some embodiments, any suitable hydrophobic acid or a combinationthereof may form a HIP pair with the conjugate of the present invention.In some embodiments, the hydrophobic acid may be a carboxylic acid (suchas but not limited to a monocarboxylic acid, dicarboxylic acid,tricarboxylic acid), a sulfinic acid, a sulfenic acid, or a sulfonicacid. In some embodiments, a salt of a suitable hydrophobic acid or acombination thereof may be used to form a HIP pair with the conjugate ofthe present invention. Examples of hydrophobic acids, saturated fattyacids, unsaturated fatty acids, aromatic acids, bile acid,polyelectrolyte, their dissociation constant in water (pKa) and log Pvalues were disclosed in WO2014/043,625, the contents of which areincorporated herein by reference in their entirety. The strength of thehydrophobic acid, the difference between the pKa of the hydrophobic acidand the pKa of the conjugate of the present invention, log P of thehydrophobic acid, the phase transition temperature of the hydrophobicacid, the molar ratio of the hydrophobic acid to the conjugate of thepresent invention, and the concentration of the hydrophobic acid werealso disclosed in WO2014/043,625, the contents of which are incorporatedherein by reference in their entirety.

In some embodiments, particles of the present invention comprising a HIPcomplex and/or prepared by a process that provides a counterion to formHIP complex with the conjugate may have a higher drug loading thanparticles without a HIP complex or prepared by a process that does notprovide any counterion to form HIP complex with the conjugate. In someembodiments, drug loading may increase 50%, 100%, 2 times, 3 times, 4times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.

In some embodiments, the particles of the invention may retain theconjugate for at least about 1 minute, at least about 15 minutes, atleast about 1 hour, when placed in a phosphate buffer solution at 37° C.

In some embodiments, the weight percentage of the conjugate in theparticles is at least about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%; 40%, 45%, or 50% such that the sum of the weightpercentages of the components of the particles is 100%. In someembodiments, the weight percentage of the conjugate in the particles isfrom about 0.5% to about 10%, or about 10% to about 20%, or about 20% toabout 30%, or about 30% to about 40%, or about 40% to about 50%, orabout 50% to about 60%, or about 60% to about 70%, or about 70% to about80%, or about 80% to about 90%, or about 90% to about 99% such that thesum of the weight percentages of the components of the particles is100%.

In some instances, a conjugate may have a molecular weight of less thanabout 50,000 Da, less than about 40,000 Da, less than about 30,000 Da,less than about 20,000 Da, less than about 15,000 Da, less than about10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or lessthan about 3,000 Da. In some cases, the conjugate may have a molecularweight of between about 1,000 Da and about 50,000 Da, between about1,000 Da and about 40,000 Da, in some embodiments between about 1,000 Daand about 30,000 Da, in some embodiments bout 1,000 Da and about 50,000Da, between about 1,000 Da and about 20,000 Da, in some embodimentsbetween about 1,000 Da and about 15,000 Da, in some embodiments betweenabout 1,000 Da and about 10,000 Da, in some embodiments between about1,000 Da and about 8,000 Da, in some embodiments between about 1,000 Daand about 5,000 Da, and in some embodiments between about 1,000 Da andabout 3,000 Da. The molecular weight of the conjugate may be calculatedas the sum of the atomic weight of each atom in the formula of theconjugate multiplied by the number of each atom. It may also be measuredby mass spectrometry, NMR, chromatography, light scattering, viscosity,and/or any other methods known in the art. It is known in the art thatthe unit of molecular weight may be g/mol, Dalton (Da), or atomic massunit (amu), wherein 1 g/mol=1 Da=1 amu.

B. Polymers

The particles may contain one or more polymers. Polymers may contain onemore of the following polyesters: homopolymers including glycolic acidunits, referred to herein as “PGA”, and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA”, and caprolactone units, such aspoly(ε-caprolactone), collectively referred to herein as “PCL”; andcopolymers including lactic acid and glycolic acid units, such asvarious forms of poly(lactic acid-co-glycolic acid) andpoly(lactide-co-glycolide) characterized by the ratio of lacticacid:glycolic acid, collectively referred to herein as “PLGA”; andpolyacrylates, and derivatives thereof. Exemplary polymers also includecopolymers of polyethylene glycol (PEG) and the aforementionedpolyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers,collectively referred to herein as “PEGylated polymers”. In certainembodiments, the PEG region can be covalently associated with polymer toyield “PEGylated polymers” by a cleavable linker.

The particles may contain one or more hydrophilic polymers. Hydrophilicpolymers include cellulosic polymers such as starch and polysaccharides;hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid(PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, orpoly-L-lysine; polyalkylene glycols and polyalkylene oxides such aspolyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethyleneoxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol);polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide);poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids);poly(vinyl alcohol); polyoxazoline; and copolymers thereof.

The particles may contain one or more hydrophobic polymers. Examples ofsuitable hydrophobic polymers include polyhydroxyacids such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacids); polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);polycarbonates such as tyrosine polycarbonates; polyamides (includingsynthetic and natural polyamides), polypeptides, and poly(amino acids);polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophobic polyethers; polyurethanes; polyetheresters; polyacetal s;polycyanoacrylates; polyacrylates; polymethylmethacrylates;polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers;polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), as well as copolymersthereof.

In certain embodiments, the hydrophobic polymer is an aliphaticpolyester. In some embodiments, the hydrophobic polymer is poly(lacticacid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid).

The particles can contain one or more biodegradable polymers.Biodegradable polymers can include polymers that are insoluble orsparingly soluble in water that are converted chemically orenzymatically in the body into water-soluble materials. Biodegradablepolymers can include soluble polymers crosslinked by hydrolyzablecross-linking groups to render the crosslinked polymer insoluble orsparingly soluble in water.

Biodegradable polymers in the particle can include polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof,alkyl cellulose such as methyl cellulose and ethyl cellulose,hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers,cellulose esters, nitro celluloses, cellulose acetate, cellulosepropionate, cellulose acetate butyrate, cellulose acetate phthalate,carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodiumsalt, polymers of acrylic and methacrylic esters such as poly (methylmethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene and polyvinylpryrrolidone, derivatives thereof,linear and branched copolymers and block copolymers thereof, and blendsthereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethylene imines), poly(caprolactones),poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides,poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates,polyphosphate esters, polyphosphazenes, derivatives thereof, linear andbranched copolymers and block copolymers thereof, and blends thereof. Insome embodiments the particle contains biodegradable polyesters orpolyanhydrides such as poly(lactic acid), poly(glycolic acid), andpoly(lactic-co-glycolic acid).

The particles can contain one or more amphiphilic polymers. Amphiphilicpolymers can be polymers containing a hydrophobic polymer block and ahydrophilic polymer block. The hydrophobic polymer, block can containone or more of the hydrophobic polymers above or a derivative orcopolymer thereof. The hydrophilic polymer block can contain one or moreof the hydrophilic polymers above or a derivative or copolymer thereof.In some embodiments the amphiphilic polymer is a di-block polymercontaining a hydrophobic end formed from a hydrophobic polymer and ahydrophilic end formed of a hydrophilic polymer. In some embodiments, amoiety can be attached to the hydrophobic end, to the hydrophilic end,or both. The particle can contain two or more amphiphilic polymers.

C. Lipids

The particles may contain one or more lipids or amphiphilic compounds.For example, the particles can be liposomes, lipid micelles, solid lipidparticles, or lipid-stabilized polymeric particles. The lipid particlecan be made from one or a mixture of different lipids. Lipid particlesare formed from one or more lipids, which can be neutral, anionic, orcationic at physiologic pH. The lipid particle, in some embodiments,incorporates one or more biocompatible lipids. The lipid particles maybe formed using a combination of more than one lipid. For example, acharged lipid may be combined with a lipid that is non-ionic oruncharged at physiological pH.

The particle can be a lipid micelle. Lipid micelles for drug deliveryare known in the art. Lipid micelles can be formed, for instance, as awater-in-oil emulsion with a lipid surfactant. An emulsion is a blend oftwo immiscible phases wherein a surfactant is added to stabilize thedispersed droplets. In some embodiments the lipid micelle is amicroemulsion. A microemulsion is a thermodynamically stable systemcomposed of at least water, oil and a lipid surfactant producing atransparent and thermodynamically stable system whose droplet size isless than 1 micron, from about 10 nm to about 500 nm, or from about 10nm to about 250 nm. Lipid micelles are generally useful forencapsulating hydrophobic active agents, including hydrophobictherapeutic agents, hydrophobic prophylactic agents, or hydrophobicdiagnostic agents.

The particle can be a liposome. Liposomes are small vesicles composed ofan aqueous medium surrounded by lipids arranged in spherical bilayers.Liposomes can be classified as small unilamellar vesicles, largeunilamellar vesicles, or multi-lamellar vesicles. Multi-lamellarliposomes contain multiple concentric lipid bilayers. Liposomes can beused to encapsulate agents, by trapping hydrophilic agents in theaqueous interior or between bilayers, or by trapping hydrophobic agentswithin the bilayer.

The lipid micelles and liposomes typically have an aqueous center. Theaqueous center can contain water or a mixture of water and alcohol.Suitable alcohols include, but are not limited to, methanol, ethanol,propanol, (such as isopropanol), butanol (such as n-butanol, isobutanol,sec-butanol, tert-butanol, pentanol (such as amyl alcohol, isobutylcarbinol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol(such as 1-heptanol, 2-heptanol, 3-heptanol and 4-heptanol) or octanol(such as 1-octanol) or a combination thereof.

The particle can be a solid lipid particle. Solid lipid particlespresent an alternative to the colloidal micelles and liposomes. Solidlipid particles are typically submicron in size, i.e from about 10 nm toabout 1 micron, from 10 nm to about 500 nm, or from 10 nm to about 250nm. Solid lipid particles are formed of lipids that are solids at roomtemperature. They are derived from oil-in-water emulsions, by replacingthe liquid oil by a solid lipid.

Suitable neutral and anionic lipids include, but are not limited to,sterols and lipids such as cholesterol, phospholipids, lysolipids,lysophospholipids, sphingolipids or pegylated lipids. Neutral andanionic lipids include, but are not limited to, phosphatidylcholine (PC)(such as egg PC, soy PC), including1,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS),phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;sphingophospholipids such as sphingomyelin and sphingoglycolipids (alsoknown as 1-ceramidyl glucosides) such as ceramide galactopyranoside,gangliosides and cerebrosides; fatty acids, sterols, containing acarboxylic acid group for example, cholesterol;1,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limitedto, 1,2-dioleylphosphoethanolamine (DOPE),1,2-dihexadecylphosphoethanolamine (DHPE),1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine(DMPC). The lipids can also include various natural (e.g., tissuederived L-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/orsynthetic (e.g., saturated and unsaturated1,2-diacyl-sn-glycero-3-phosphocholines,1-acyl-2-acyl-sn-glycero-3-phosphocholines,1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids.

Suitable cationic lipids include, but are not limited to,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, alsoreferences as TAP lipids, for example methylsulfate salt. Suitable TAPlipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP(dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitablecationic lipids in the liposomes include, but are not limited to,dimethyldioctadecyl ammonium bromide (DDAB),1,2-diacyloxy-3-trimethylammonium propanes,N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP),1,2-diacyloxy-3-dimethylammonium propanes,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine (DOGS),3-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyl trimethylammonium bromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N, N,N′, N′-tetramethyl-,N-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide. Inone embodiment, the cationic lipids can be1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, for example,1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM), and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM). In one embodiment, the cationic lipids can be2,3-dialkyloxypropyl quaternary ammonium compound derivatives containinga hydroxyalkyl moiety on the quaternary amine, for example,1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide(DORIE-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxy ethylammonium bromide (DSRIE).

Suitable solid lipids include, but are not limited to, higher saturatedalcohols, higher fatty acids, sphingolipids, synthetic esters, andmono-, di-, and triglycerides of higher saturated fatty acids. Solidlipids can include aliphatic alcohols having 10-40, for example, 12-30carbon atoms, such as cetostearyl alcohol. Solid lipids can includehigher fatty acids of 10-40, for example, 12-30 carbon atoms, such asstearic acid, palmitic acid, decanoic acid, and behenic acid. Solidlipids can include glycerides, including monoglycerides, diglycerides,and triglycerides, of higher saturated fatty acids having 10-40, forexample, 12-30 carbon atoms, such as glyceryl monostearate, glycerolbehenate, glycerol palmitostearate, glycerol trilaurate, tricaprin,trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castoroil. Suitable solid lipids can include cetyl palmitate, beeswax, orcyclodextrin.

Amphiphilic compounds include, but are not limited to, phospholipids,such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), for example, between 0.1-30(weight lipid/w polymer). Phospholipids that may be used include, butare not limited to, phosphatidic acids, phosphatidyl cholines with bothsaturated and unsaturated lipids, phosphatidyl ethanolamines,phosphatidylglycerols, phosphatidylserines, phosphatidylinositols,lysophosphatidyl derivatives, cardiolipin, and β-acyl-y-alkylphospholipids. Examples of phospholipids include, but are not limitedto, phosphatidylcholines such as dioleoylphosphatidylcholine,dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholinedilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine(DAPC), dibehenoylphosphatidylcho-line (DBPC),ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine(DLPC); and phosphatidylethanolamines such asdioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophosphoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

D. Additional Active Agents

The particles can contain one or more additional active agents inaddition to those in the conjugates. The additional active agents can betherapeutic, prophylactic, diagnostic, or nutritional agents as listedabove. The additional active agents can be present in any amount, e.g.from about 0.5% to about 90%, from about 0.5% to about 50%, from about0.5% to about 25%, from about 0.5% to about 20%, from about 0.5% toabout 10%, or from about 5% to about 10% (w/w) based upon the weight ofthe particle. In one embodiment, the agents are incorporated in an about0.5% to about 10% loading w/w.

E. Additional Targeting Moieties

The particles can contain one or more targeting moieties targeting theparticle to a specific organ, tissue, cell type, or subcellularcompartment in addition to the targeting moieties of the conjugate. Theadditional targeting moieties can be present on the surface of theparticle, on the interior of the particle, or both. The additionaltargeting moieties can be immobilized on the surface of the particle,e.g., can be covalently attached to polymer or lipid in the particle. Insome embodiments, the additional targeting moieties are covalentlyattached to an amphiphilic polymer or a lipid such that the targetingmoieties are oriented on the surface of the particle.

IV. Formulations

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the conjugate orparticles comprising the conjugates to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

The conjugates or particles of the present invention can be formulatedusing one or more excipients to: (1) increase stability; (2) permit thesustained or delayed release (e.g., from a depot formulation of themonomaleimide); (3) alter the biodistribution (e.g., target themonomaleimide compounds to specific tissues or cell types); (4) alterthe release profile of the monomaleimide compounds in vivo. Non-limitingexamples of the excipients include any and all solvents, dispersionmedia, diluents, or other liquid vehicles, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, and preservatives. Excipients of the present invention may alsoinclude, without limitation, lipidoids, liposomes, lipid nanoparticles,polymers, lipoplexes, core-shell nanoparticles, peptides, proteins,hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the invention may include one or moreexcipients, each in an amount that together increases the stability ofthe monomaleimide compounds.

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21st Edition,A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference in its entirety) discloses variousexcipients used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional excipient medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEENn® 60],polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate[SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate[SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC® F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, chamomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Administration

The conjugates or particles of the present invention may be administeredby any route which results in a therapeutically effective outcome. Theseinclude, but are not limited to enteral, gastroenteral, epidural, oral,transdermal, epidural (peridural), intracerebral (into the cerebrum),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intraarterial (into an artery), intramuscular(into a muscle), intracardiac (into the heart), intraosseous infusion(into the bone marrow), intrathecal (into the spinal canal),intraperitoneal, (infusion or injection into the peritoneum),intravesical infusion, intravitreal, (through the eye), intracavernousinjection, (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), insufflation (snorting),sublingual, sublabial, enema, eye drops (onto the conjunctiva), or inear drops. In specific embodiments, compositions may be administered ina way which allows them to cross the blood-brain barrier, vascularbarrier, or other epithelial barrier.

The formulations described herein contain an effective amount ofconjugates or particles in a pharmaceutical carrier appropriate foradministration to an individual in need thereof. The formulations may beadministered parenterally (e.g., by injection or infusion). Theformulations or variations thereof may be administered in any mannerincluding enterally, topically (e.g., to the eye), or via pulmonaryadministration. In some embodiments the formulations are administeredtopically.

A. Parenteral Formulations

The particles can be formulated for parenteral delivery, such asinjection or infusion, in the form of a solution, suspension oremulsion. The formulation can be administered systemically, regionallyor directly to the organ or tissue to be treated.

Parenteral formulations can be prepared as aqueous compositions usingtechniques is known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In some cases, an isotonic agent is included, for example,one or more sugars, sodium chloride, or other suitable agent known inthe art.

Solutions and dispersions of the particles can be prepared in water oranother solvent or dispersing medium suitably mixed with one or morepharmaceutically acceptable excipients including, but not limited to,surfactants, dispersants, emulsifiers, pH modifying agents, andcombinations thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s) or particles.

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.If using 10% sucrose or 5% dextrose, a buffer may not be required.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating theparticles in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized particles into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions,examples of methods of preparation include vacuum-drying andfreeze-drying techniques that yield a powder of the particle plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are known in the art.

Pharmaceutical formulations for parenteral administration can be in theform of a sterile aqueous solution or suspension of particles formedfrom one or more polymer-drug conjugates. Acceptable solvents include,for example, water, Ringer's solution, phosphate buffered saline (PBS),and isotonic sodium chloride solution. The formulation may also be asterile solution, suspension, or emulsion in a nontoxic, parenterallyacceptable diluent or solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for parenteral administrationcan be packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for parenteral administration maybe buffered with an effective amount of buffer necessary to maintain apH suitable for ocular administration. Suitable buffers are well knownby those skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more tonicity agents to adjust the isotonic range ofthe formulation. Suitable tonicity agents are well known in the art andsome examples include glycerin, sucrose, dextrose, mannitol, sorbitol,sodium chloride, and other electrolytes.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more preservatives to prevent bacterialcontamination of the ophthalmic preparations. Suitable preservatives areknown in the art, and include polyhexamethylenebiguanidine (PHMB),benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwiseknown as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more excipients known art, such as dispersingagents, wetting agents, and suspending agents.

B. Mucosal Topical Formulations

The particles can be formulated for topical administration to a mucosalsurface Suitable dosage forms for topical administration include creams,ointments, salves, sprays, gels, lotions, emulsions, liquids, andtransdermal patches. The formulation may be formulated for transmucosaltransepithelial, or transendothelial administration. The compositionscontain one or more chemical penetration enhancers, membranepermeability agents, membrane transport agents, emollients, surfactants,stabilizers, and combination thereof. In some embodiments, the particlescan be administered as a liquid formulation, such as a solution orsuspension, a semi-solid formulation, such as a lotion or ointment, or asolid formulation. In some embodiments, the particles are formulated asliquids, including solutions and suspensions, such as eye drops or as asemi-solid formulation, to the mucosa, such as the eye or vaginally orrectally.

“Surfactants” are surface-active agents that lower surface tension andthereby increase the emulsifying, foaming, dispersing, spreading andwetting properties of a product. Suitable non-ionic surfactants includeemulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters,benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate,poloxamer, povidone and combinations thereof. In one embodiment, thenon-ionic surfactant is stearyl alcohol.

“Emulsifiers” are surface active substances which promote the suspensionof one liquid in another and promote the formation of a stable mixture,or emulsion, of oil and water. Common emulsifiers are: metallic soaps,certain animal and vegetable oils, and various polar compounds. Suitableemulsifiers include acacia, anionic emulsifying wax, calcium stearate,carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol,diethanolamine, ethylene glycol palmitostearate, glycerin monostearate,glyceryl monooleate, hydroxypropyl cellulose, hypromellose, lanolin,hydrous, lanolin alcohols, lecithin, medium-chain triglycerides,methylcellulose, mineral oil and lanolin alcohols, monobasic sodiumphosphate, monoethanolamine, nonionic emulsifying wax, oleic acid,poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylenecastor oil derivatives, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, propylene glycol alginate, self-emulsifyingglyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate,sorbitan esters, stearic acid, sunflower oil, tragacanth,triethanolamine, xanthan gum and combinations thereof. In oneembodiment, the emulsifier is glycerol stearate.

Suitable classes of penetration enhancers are known in the art andinclude, but are not limited to, fatty alcohols, fatty acid esters,fatty acids, fatty alcohol ethers, amino acids, phospholipids,lecithins, cholate salts, enzymes, amines and amides, complexing agents(liposomes, cyclodextrins, modified celluloses, and diimides),macrocyclics, such as macrocylic lactones, ketones, and anhydrides andcyclic ureas, surfactants, N-methyl pyrrolidones and derivativesthereof, DMSO and related compounds, ionic compounds, azone and relatedcompounds, and solvents, such as alcohols, ketones, amides, polyols(e.g., glycols). Examples of these classes are known in the art.

Dosing

The present invention provides methods comprising administeringconjugates or particles containing the conjugate as described herein toa subject in need thereof. Conjugates or particles containing theconjugates as described herein may be administered to a subject usingany amount and any route of administration effective for preventing ortreating or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like.

Compositions in accordance with the invention are typically formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage of thecompositions of the present invention may be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or appropriateimaging dose level for any particular patient will depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In some embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In some embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

As used herein, a “split dose” is the division of single unit dose ortotal daily dose into two or more doses, e.g, two or moreadministrations of the single unit dose. As used herein, a “single unitdose” is a dose of any therapeutic administered in one dose/at onetime/single route/single point of contact, i.e., single administrationevent. As used herein, a “total daily dose” is an amount given orprescribed in 24 hr period. It may be administered as a single unitdose. In one embodiment, the monomaleimide compounds of the presentinvention are administered to a subject in split doses. Themonomaleimide compounds may be formulated in buffer only or in aformulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the monomaleimidecompounds then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered monomaleimide compound may beaccomplished by dissolving or suspending the monomalimide in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the monomaleimide compounds in biodegradable polymers suchas polylactide-polyglycolide. Depending upon the ratio of monomaleimidecompounds to polymer and the nature of the particular polymer employed,the rate of monomaleimide compound release can be controlled. Examplesof other biodegradable polymers include, but are not limited to,poly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay be prepared by entrapping the monomaleimide compounds in liposomesor microemulsions which are compatible with body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be used for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 um to 500 um. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

V. Methods of Making Particles

In various embodiments, a method of making the particles includesproviding a conjugate; providing a base component such as PLA-PEG orPLGA-PEG for forming a particle; combining the conjugate and the basecomponent in an organic solution to form a first organic phase; andcombining the first organic phase with a first aqueous solution to forma second phase; emulsifying the second phase to form an emulsion phase;and recovering particles. In various embodiments, the emulsion phase isfurther homogenized. In some embodiments, the first phase includes about5 to about 50% weight, e.g. about 1 to about 40% solids, or about 5 toabout 30% solids, e.g. about 5%, 10%, 15%, and 20%, of the conjugate andthe base component. In certain embodiments, the first phase includesabout 5% weight of the conjugate and the base component. In variousembodiments, the organic phase comprises acetonitrile, tetrahydrofuran,ethyl acetate, isopropyl alcohol, isopropyl acetate, dimethylformamide,methylene chloride, dichloromethane, chloroform, acetone, benzylalcohol, TWEEN® 80, SPAN® 80, or a combination thereof. In someembodiments, the organic phase includes benzyl alcohol, ethyl acetate,or a combination thereof.

In various embodiments, the aqueous solution includes water, sodiumcholate, ethyl acetate, or benzyl alcohol. In various embodiments, asurfactant is added into the first phase, the second phase, or both. Asurfactant, in some instances, can act as an emulsifier or a stabilizerfor a composition disclosed herein. A suitable surfactant can be acationic surfactant, an anionic surfactant, or a nonionic surfactant. Insome embodiments, a surfactant suitable for making a compositiondescribed herein includes sorbitan fatty acid esters, polyoxyethylenesorbitan fatty acid esters and polyoxyethylene stearates. Examples ofsuch fatty acid ester nonionic surfactants are the TWEEN® 80, SPAN® 80,and MYJ® surfactants from ICI. SPAN® surfactants include C₁₂-C₁₈sorbitan monoesters. TWEEN® surfactants include poly(ethylene oxide)C₁₂-C₁₈ sorbitan monoesters. MYJ® surfactants include poly(ethyleneoxide) stearates. In certain embodiments, the aqueous solution alsocomprises a surfactant (e.g., an emulsifier), including a polysorbate.For example, the aqueous solution can include polysorbate 80. In someembodiments, a suitable surfactant includes a lipid-based surfactant.For example, the composition can include1,2-dihexanoyl-sn-glycero-3-phosphocholine,1,2-diheptanoyl-sn-glycero-3-phosphocholine, PEGlyated1,2-distearoyl-sn-glycero-3-phosphoethanolamine (includingPEG5000-DSPE), PEGlyated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(including1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt)).

Emulsifying the second phase to form an emulsion phase may be performedin one or two emulsification steps. For example, a primary emulsion maybe prepared, and then emulsified to form a fine emulsion. The primaryemulsion can be formed, for example, using simple mixing, a highpressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g. a probe sonicator or a high pressurehomogenizer, e.g. by pass(es) through a homogenizer. For example, when ahigh pressure homogenizer is used, the pressure used may be about 4000to about 8000 psi, about 4000 to about 5000 psi, or 4000 or 5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. Quenching may beperformed at least partially at a temperature of about 5° C. or less.For example, water used in the quenching may be at a temperature that isless that room temperature (e.g. about 0 to about 10° C., or about 0 toabout 5° C.).

In various embodiments, the particles are recovered by filtration. Forexample, ultrafiltration membranes can be used. Exemplary filtration maybe performed using a tangential flow filtration system. For example, byusing a membrane with a pore size suitable to retain particles whileallowing solutes, micelles, and organic solvent to pass, particles canbe selectively separated. Exemplary membranes with molecular weightcut-offs of about 300-500 kDa (−5-25 nm) may be, used.

In various embodiments, the particles are freeze-dried or lyophilized,in some instances, to extend their shelf life. In some embodiments, thecomposition also includes a lyoprotectant. In certain embodiments, alyoprotectant is selected from a sugar, a polyalcohol, or a derivativethereof. In some embodiments, a lyoprotectant is selected from amonosaccharide, a disaccharide, or a mixture thereof. For example, alyoprotectant can be sucrose, lactulose, trehalose, lactose, glucose,maltose, mannitol, cellobiose, or a mixture thereof.

Methods of making particles containing one or more conjugates areprovided. The particles can be polymeric particles, lipid particles, orcombinations thereof. The various methods described herein can beadjusted to control the size and composition of the particles, e.g. somemethods are best suited for preparing microparticles while others arebetter suited for preparing particles. The selection of a method forpreparing particles having the descried characteristics can be performedby the skilled artisan without undue experimentation.

i. Polymeric Particles

Methods of making polymeric particles are known in the art. Polymericparticles can be prepared using any suitable method known in the art.Common microencapsulation techniques include, but are not limited to,spray drying, interfacial polymerization, hot melt encapsulation, phaseseparation encapsulation (spontaneous emulsion microencapsulation,solvent evaporation microencapsulation, and solvent removalmicroencapsulation), coacervation, low temperature microsphereformation, and phase inversion nanoencapsulation (PIN). A brief summaryof these methods is presented below.

1. Spray Drying

Methods for forming polymeric particles using spray drying techniquesare described in U.S. Pat. No. 6,620,617. In this method, the polymer isdissolved in an organic solvent such as methylene chloride or in water.A known amount of one or more conjugates or additional active agents tobe incorporated in the particles is suspended (in the case of aninsoluble active agent) or co-dissolved (in the case of a soluble activeagent) in the polymer solution. The solution or dispersion is pumpedthrough a micronizing nozzle driven by a flow of compressed gas, and theresulting aerosol is suspended in a heated cyclone of air, allowing thesolvent to evaporate from the microdroplets, forming particles.Microspheres/nanospheres ranging between 0.1 10 microns can be obtainedusing this method.

2. Interfacial Polymerization

Interfacial polymerization can also be used to encapsulate one or moreconjugates and/or active agents. Using this method, a monomer and theconjugates or active agent(s) are dissolved in a solvent. A secondmonomer is dissolved in a second solvent (typically aqueous) which isimmiscible with the first. An emulsion is formed by suspending the firstsolution through stirring in the second solution. Once the emulsion isstabilized, an initiator is added to the aqueous phase causinginterfacial polymerization at the interface of each droplet of emulsion.

3. Hot Melt Microencapsulation

Microspheres can be formed from polymers such as polyesters andpolyanhydrides using hot melt microencapsulation methods as described inMathiowitz et al., Reactive Polymers, 6:275 (1987). In some embodimentsemploying this method, polymers with molecular weights between3,000-75,000 daltons are used. In this method, the polymer first ismelted and then mixed with the solid particles of one or more activeagents to be incorporated that have been sieved to less than 50 microns.The mixture is suspended in a non-miscible solvent (like silicon oil),and, with continuous stirring, heated to 5° C. above the melting pointof the polymer. Once the emulsion is stabilized, it is cooled until thepolymer particles solidify. The resulting microspheres are washed bydecanting with petroleum ether to produce a free flowing powder.

4. Phase Separation Microencapsulation

In phase separation microencapsulation techniques, a polymer solution isstirred, optionally in the presence of one or more active agents to beencapsulated. While continuing to uniformly suspend the material throughstirring, a nonsolvent for the polymer is slowly added to the solutionto decrease the polymer's solubility. Depending on the solubility of thepolymer in the solvent and nonsolvent, the polymer either precipitatesor phase separates into a polymer rich and a polymer poor phase. Underproper conditions, the polymer in the polymer rich phase will migrate tothe interface with the continuous phase, encapsulating the activeagent(s) in a droplet with an outer polymer shell.

a. Spontaneous Emulsion Microencapsulation

Spontaneous emulsification involves solidifying emulsified liquidpolymer droplets formed above by changing temperature, evaporatingsolvent, or adding chemical cross-linking agents. The physical andchemical properties of the encapsulant, as well as the properties of theone or more active agents optionally incorporated into the nascentparticles, dictates suitable methods of encapsulation. Factors such ashydrophobicity, molecular weight, chemical stability, and thermalstability affect encapsulation.

b. Solvent Evaporation Microencapsulation

Methods for forming microspheres using solvent evaporation techniquesare described in Mathiowitz et al., J. Scanning Microscopy, 4:329(1990); Beck et al., Fertil. Steril., 31:545 (1979); Beck et al., Am. J.Obstet. Gynecol. 135(3) (1979); Benita et al., J. Pharm. Sci., 73:1721(1984); and U.S. Pat. No. 3,960,757. The polymer is dissolved in avolatile organic solvent, such as methylene chloride. One or more activeagents to be incorporated are optionally added to the solution, and themixture is suspended in an aqueous solution that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporated, leaving solidmicroparticles/nanoparticles. This method is useful for relativelystable polymers like polyesters and polystyrene.

c. Solvent Removal Microencapsulation

The solvent removal microencapsulation technique is primarily designedfor polyanhydrides and is described, for example, in WO 93/21906. Inthis method, the substance to be incorporated is dispersed or dissolvedin a solution of the selected polymer in a volatile organic solvent,such as methylene chloride. This mixture is suspended by stirring in anorganic oil, such as silicon oil, to form an emulsion. Microspheres thatrange between 1-300 microns can be obtained by this procedure.Substances which can be incorporated in the microspheres includepharmaceuticals, pesticides, nutrients, imaging agents, and metalcompounds.

5. Coacervation

Encapsulation procedures for various substances using coacervationtechniques are known in the art, for example, in GB-B-929 406; GB-B-92940 1; and U.S. Pat. Nos. 3,266,987, 4,794,000, and 4,460,563.Coacervation involves the separation of a macromolecular solution intotwo immiscible liquid phases. One phase is a dense coacervate phase,which contains a high concentration of the polymer encapsulant (andoptionally one or more active agents), while the second phase contains alow concentration of the polymer. Within the dense coacervate phase, thepolymer encapsulant forms nanoscale or microscale droplets. Coacervationmay be induced by a temperature change, addition of a non-solvent oraddition of a micro-salt (simple coacervation), or by the addition ofanother polymer thereby forming an interpolymer complex (complexcoacervation).

6. Low Temperature Casting of Microspheres

Methods for very low temperature casting of controlled release particlesare described in U.S. Pat. No. 5,019,400. In this method, a polymer isdissolved in a solvent optionally with one or more dissolved ordispersed active agents. The mixture is then atomized into a vesselcontaining a liquid non-solvent at a temperature below the freezingpoint of the polymer substance solution which freezes the polymerdroplets. As the droplets and non-solvent for the polymer are warmed,the solvent in the droplets thaws and is extracted into the non-solvent,resulting in the hardening of the microspheres.

7. Phase Inversion Nanoencapsulation (PIN)

Particles can also be formed using the phase inversion nanoencapsulation(PIN) method, wherein a polymer is dissolved in a “good” solvent, fineparticles of a substance to be incorporated, such as a drug, are mixedor dissolved in the polymer solution, and the mixture is poured into astrong non-solvent for the polymer, to spontaneously produce, underfavorable conditions, polymeric microspheres, wherein the polymer iseither coated with the particles or the particles are dispersed in thepolymer. See, e.g., U.S. Pat. No. 6,143,211. The method can be used toproduce monodisperse populations of nanoparticles and microparticles ina wide range of sizes, including, for example, about 100 nanometers toabout 10 microns.

Advantageously, an emulsion need not be formed prior to precipitation.The process can be used to form microspheres from thermoplasticpolymers.

8. Emulsion Methods

In some embodiments, a particle is prepared using an emulsion solventevaporation method. For example, a polymeric material is dissolved in awater immiscible organic solvent and mixed with a drug solution or acombination of drug solutions. In some embodiments a solution of atherapeutic, prophylactic, or diagnostic agent to be encapsulated ismixed with the polymer solution. The polymer can be, but is not limitedto, one or more of the following: PLA, PGA, PCL, their copolymers,polyacrylates, the aforementioned PEGylated polymers. The drug moleculescan include one or more conjugates as described above and one or moreadditional active agents. The water immiscible organic solvent, can be,but is not limited to, one or more of the following: chloroform,dichloromethane, and acyl acetate. The drug can be dissolved in, but isnot limited to, one or more of the following: acetone, ethanol,methanol, isopropyl alcohol, acetonitrile and Dimethyl sulfoxide (DMSO).

An aqueous solution is added into the resulting polymer solution toyield emulsion solution by emulsification. The emulsification techniquecan be, but not limited to, probe sonication or homogenization through ahomogenizer.

9. Nanoprecipitation

In another embodiment, a conjugate containing nanoparticle is preparedusing nanoprecipitation methods or microfluidic devices. The conjugatecontaining polymeric material is mixed with a drug or drug combinationsin a water miscible organic solvent, optionally containing additionalpolymers. The additional polymer can be, but is not limited to, one ormore of the following: PLA, PGA, PCL, their copolymers, polyacrylates,the aforementioned PEGylated polymers. The water miscible organicsolvent, can be, but is not limited to, one or more of the following:acetone, ethanol, methanol, isopropyl alcohol, acetonitrile and dimethylsulfoxide (DMSO). The resulting mixture solution is then added to apolymer non-solvent, such as an aqueous solution, to yield nanoparticlesolution.

10. Microfluidics

Methods of making particles using microfluidics are known in the art.Suitable methods include those described in U.S. Patent ApplicationPublication No. 2010/0022680 A1. In general, the microfluidic devicecomprises at least two channels that converge into a mixing apparatus.The channels are typically formed by lithography, etching, embossing, ormolding of a polymeric surface. A source of fluid is attached to eachchannel, and the application of pressure to the source causes the flowof the fluid in the channel. The pressure may be applied by a syringe, apump, and/or gravity. The inlet streams of solutions with polymer,targeting moieties, lipids, drug, payload, etc. converge and mix, andthe resulting mixture is combined with a polymer non-solvent solution toform the particles having the desired size and density of moieties onthe surface. By varying the pressure and flow rate in the inlet channelsand the nature and composition of the fluid sources particles can beproduced having reproducible size and structure.

ii. Lipid Particles

Methods of making lipid particles are known in the art. Lipid particlescan be lipid micelles, liposomes, or solid lipid particles preparedusing any suitable method known in the art. Common techniques forcreated lipid particles encapsulating an active agent include, but arenot limited to high pressure homogenization techniques, supercriticalfluid methods, emulsion methods, solvent diffusion methods, and spraydrying. A brief summary of these methods is presented below.

1. High Pressure Homogenization (HPH) Methods

High pressure homogenization is a reliable and powerful technique, whichis used for the production of smaller lipid particles with narrow sizedistributions, including lipid micelles, liposomes, and solid lipidparticles. High pressure homogenizers push a liquid with high pressure(100-2000 bar) through a narrow gap (in the range of a few microns). Thefluid can contain lipids that are liquid at room temperature or a meltof lipids that are solid at room temperature. The fluid accelerates on avery short distance to very high velocity (over 1000 Km/h). This createshigh shear stress and cavitation forces that disrupt the particles,generally down to the submicron range. Generally, 5-10% lipid content isused but up to 40% lipid content has also been investigated.

Two approaches of HPH are hot homogenization and cold homogenization,work on the same concept of mixing the drug in bulk of lipid solution ormelt.

a. Hot homogenization:

Hot homogenization is carried out at temperatures above the meltingpoint of the lipid and can therefore be regarded as the homogenizationof an emulsion. A pre-emulsion of the drug loaded lipid melt and theaqueous emulsifier phase is obtained by a high-shear mixing. HPH of thepre-emulsion is carried out at temperatures above the melting point ofthe lipid. A number of parameters, including the temperature, pressure,and number of cycles, can be adjusted to produce lipid particles withthe desired size. In general, higher temperatures result in lowerparticle sizes due to the decreased viscosity of the inner phase.However, high temperatures increase the degradation rate of the drug andthe carrier. Increasing the homogenization pressure or the number ofcycles often results in an increase of the particle size due to highkinetic energy of the particles.

b. Cold Homogenization

Cold homogenization has been developed as an alternative to hothomogenization. Cold homogenization does not suffer from problems suchas temperature-induced drug degradation or drug distribution into theaqueous phase during homogenization. The cold homogenization isparticularly useful for solid lipid particles, but can be applied withslight modifications to produce liposomes and lipid micelles. In thistechnique the drug containing lipid melt is cooled, the solid lipidground to lipid microparticles and these lipid microparticles aredispersed in a cold surfactant solution yielding a pre-suspension. Thepre-suspension is homogenized at or below room temperature, where thegravitation force is strong enough to break the lipid microparticlesdirectly to solid lipid nanoparticles.

2. Ultrasonication/High Speed Homogenization Methods

Lipid particles, including lipid micelles, liposomes, and solid lipidparticles, can be prepared by ultrasonication/high speed homogenization.The combination of both ultrasonication and high-speed homogenization isparticularly useful for the production of smaller lipid particles.Liposomes are formed in the size range from 10 nm to 200 nm, forexample, 50 nm to 100 nm, by this process.

3. Solvent Evaporation Methods

Lipid particles can be prepared by solvent evaporation approaches. Thelipophilic material is dissolved in a water-immiscible organic solvent(e.g. cyclohexane) that is emulsified in an aqueous phase. Uponevaporation of the solvent, particles dispersion is formed byprecipitation of the lipid in the aqueous medium. Parameters such astemperature, pressure, choices of solvents can be used to controlparticle size and distribution. Solvent evaporation rate can be adjustedthrough increased/reduced pressure or increased/reduced temperature.

4. Solvent Emulsification-Diffusion Methods

Lipid particles can be prepared by solvent emulsification-diffusionmethods. The lipid is first dissolved in an organic phase, such asethanol and acetone. An acidic aqueous phase is used to adjust the zetapotential to induce lipid coacervation. The continuous flow mode allowsthe continuous diffusion of water and alcohol, reducing lipidsolubility, which causes thermodynamic instability and generatesliposomes

5. Supercritical Fluid Methods

Lipid particles, including liposomes and solid lipid particles, can beprepared from supercritical fluid methods. Supercritical fluidapproaches have the advantage of replacing or reducing the amount of theorganic solvents used in other preparation methods. The lipids, activeagents to be encapsulated, and excipients can be solvated at highpressure in a supercritical solvent. The supercritical solvent is mostcommonly CO₂, although other supercritical solvents are known in theart. To increase solubility of the lipid, a small amount of co-solventcan be used. Ethanol is a common co-solvent, although other smallorganic solvents that are generally regarded as safe for formulationscan be used. The lipid particles, lipid micelles, liposomes, or solidlipid particles can be obtained by expansion of the supercriticalsolution or by injection into a non-solvent aqueous phase. The particleformation and size distribution can be controlled by adjusting thesupercritical solvent, co-solvent, non-solvent, temperatures, pressures,etc.

6. Microemulsion Based Methods

Microemulsion based methods for making lipid particles are known in theart. These methods are based upon the dilution of a multiphase, usuallytwo-phase, system. Emulsion methods for the production of lipidparticles generally involve the formation of a water-in-oil emulsionthrough the addition of a small amount of aqueous media to a largervolume of immiscible organic solution containing the lipid. The mixtureis agitated to disperse the aqueous media as tiny droplets throughoutthe organic solvent and the lipid aligns itself into a monolayer at theboundary between the organic and aqueous phases. The size of thedroplets is controlled by pressure, temperature, the agitation appliedand the amount of lipid present.

The water-in-oil emulsion can be transformed into a liposomal suspensionthrough the formation of a double emulsion. In a double emulsion, theorganic solution containing the water droplets is added to a largevolume of aqueous media and agitated, producing a water-in-oil-in-wateremulsion. The size and type of lipid particle formed can be controlledby the choice of and amount of lipid, temperature, pressure,co-surfactants, solvents, etc.

7. Spray Drying Methods

Spray drying methods similar to those described above for makingpolymeric particle can be employed to create solid lipid particles.Typically, this method is used with lipids with a melting point above70° C.

In some embodiments, conjugates of the present invention may beencapsulated in polymeric particles using a single oil in water emulsionmethod. As a non-limiting example, the conjugate and a suitable polymeror block copolymer or a mixture of polymers/block copolymers, aredissolved in organic solvents such as, but not limited to,dichloromethane (DCM), ethyl acetate (EtAc) or chroloform to form theoil phase. Co-solvents such as, but not limited to, dimethyl formamide(DMF), acetonitrile (CAN) or benzyl alcohol (BA) may be used to controlthe size of the particles and/or to solubilize the conjugate. Polymersused in the formulation may include, but not limited to, PLA97-b-PEG5,PLA35-b-PEG5 and PLA16-b-PEG5 copolymers.

In some embodiments, particle formulations may be prepared by varyingthe lipophilicity of conjugates of the present invention. Thelipophilicity may be varied by using hydrophobic ion-pairs orhydrophobic ion-paring (HIP) of the conjugates with differentcounterions. HIP alters the solubility of the conjugates of the presentinvention. The aqueous solubility may drop and the solubility in organicphases may increase.

Any suitable agent may be used to provide counterions to form HIPcomplex with the conjugate of the present invention. In someembodiments, the HIP complex may be formed prior to formulation of theparticles.

VI. Methods of Using the Conjugates and Particles

The conjugates or particles as described herein can be administered totreat any hyperproliferative disease, metabolic disease, infectiousdisease, or cancer, as appropriate. The formulations can be used forimmunization. Formulations may be administered by injection, orally, ortopically, typically to a mucosal surface (lung, nasal, oral, buccal,sublingual, vaginally, rectally) or to the eye (intraocularly ortransocularly).

In various embodiments, methods for treating a subject having a cancerare provided, wherein the method comprises administering atherapeutically-effective amount of the conjugates or particles, asdescribed herein, to a subject having a cancer, suspected of havingcancer, or having a predisposition to a cancer. According to the presentinvention, cancer embraces any disease or malady characterized byuncontrolled cell proliferation, e.g., hyperproliferation. Cancers maybe characterized by tumors, e.g., solid tumors or any neoplasm.

In some embodiments, the subject may be otherwise free of indicationsfor treatment with the conjugates or particles. In some embodiments,methods include use of cancer cells, including but not limited tomammalian cancer cells. In some instances, the mammalian cancer cellsare human cancer cells.

In some embodiments, the conjugates or particles of the presentteachings have been found to inhibit cancer and/or tumor growth. Theymay also reduce, including cell proliferation, invasiveness, and/ormetastasis, thereby rendering them useful for the treatment of a cancer.

In some embodiments, the conjugates or particles of the presentteachings may be used to prevent the growth of a tumor or cancer, and/orto prevent the metastasis of a tumor or cancer. In some embodiments,compositions of the present teachings may be used to shrink or destroy acancer.

In some embodiments, the conjugates or particles provided herein areuseful for inhibiting proliferation of a cancer cell. In someembodiments, the conjugates or particles provided herein are useful forinhibiting cellular proliferation, e.g., inhibiting the rate of cellularproliferation, preventing cellular proliferation, and/or inducing celldeath. In general, the conjugates or particles as described herein caninhibit cellular proliferation of a cancer cell or both inhibitingproliferation and/or inducing cell death of a cancer cell.

The cancers treatable by methods of the present teachings generallyoccur in mammals. Mammals include, for example, humans, non-humanprimates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses,pigs, sheep, goats, and cattle. In various embodiments, the cancer islung cancer, breast cancer, e.g., mutant BRCA1 and/or mutant BRCA2breast cancer, non-BRCA-associated breast cancer, colorectal cancer,ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer,prostate cancer, cervical cancer, renal cancer, leukemia, centralnervous system cancers, myeloma, and melanoma. In some embodiments, thecancer is lung cancer. In certain embodiments, the cancer is human lungcarcinoma, ovarian cancer, pancreatic cancer or colorectal cancer.

The conjugates or particles as described herein or formulationscontaining the conjugates or particles as described herein can be usedfor the selective tissue delivery of a therapeutic, prophylactic, ordiagnostic agent to an individual or patient in need thereof. Dosageregimens may be adjusted to provide the optimum desired response (e.g.,a therapeutic or prophylactic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic.

In various embodiments, a conjugate contained within a particle isreleased in a controlled manner. The release can be in vitro or in vivo.For example, particles can be subject to a release test under certainconditions, including those specified in the U.S. Pharmacopeia andvariations thereof.

In various embodiments, less than about 90%, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20% of the conjugatecontained within particles is released in the first hour after theparticles are exposed to the conditions of a release test. In someembodiments, less that about 90%, less than about 80%, less than about70%, less than about 60%, or less than about 50% of the conjugatecontained within particles is released in the first hour after theparticles are exposed to the conditions of a release test. In certainembodiments, less than about 50% of the conjugate contained withinparticles is released in the first hour after the particles are exposedto the conditions of a release test.

With respect to a conjugate being released in vivo, for instance, theconjugate contained within a particle administered to a subject may beprotected from a subject's body, and the body may also be isolated fromthe conjugate until the conjugate is released from the particle.

Thus, in some embodiments, the conjugate may be substantially containedwithin the particle until the particle is delivered into the body of asubject. For example, less than about 90%, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about15%, less than about 10%, less than about 5%, or less than about 1% ofthe total conjugate is released from the particle prior to the particlebeing delivered into the body, for example, a treatment site, of asubject. In some embodiments, the conjugate may be released over anextended period of time or by bursts (e.g., amounts of the conjugate arereleased in a short period of time, followed by a period of time wheresubstantially no conjugate is released). For example, the conjugate canbe released over 6 hours, 12 hours, 24 hours, or 48 hours. In certainembodiments, the conjugate is released over one week or one month.

VII. Kits and Devices

The invention provides a variety of kits and devices for convenientlyand/or effectively carrying out methods of the present invention.Typically, kits will comprise sufficient amounts and/or numbers ofcomponents to allow a user to perform multiple treatments of asubject(s) and/or to perform multiple experiments.

In one embodiment, the present invention provides kits for inhibitingtumor cell growth in vitro or in vivo, comprising a conjugate and/orparticle of the present invention or a combination of conjugates and/orparticles of the present invention, optionally in combination with anyother active agents.

The kit may further comprise packaging and instructions and/or adelivery agent to form a formulation composition. The delivery agent maycomprise a saline, a buffered solution, or any delivery agent disclosedherein. The amount of each component may be varied to enable consistent,reproducible higher concentration saline or simple buffer formulations.The components may also be varied in order to increase the stability ofthe conjugates and/or particles in the buffer solution over a period oftime and/or under a variety of conditions.

The present invention provides for devices which may incorporateconjugates and/or particles of the present invention. These devicescontain in a stable formulation available to be immediately delivered toa subject in need thereof, such as a human patient. In some embodiments,the subject has cancer.

Non-limiting examples of the devices include a pump, a catheter, aneedle, a transdermal patch, a pressurized olfactory delivery device,iontophoresis devices, multi-layered microfluidic devices. The devicesmay be employed to deliver conjugates and/or particles of the presentinvention according to single, multi- or split-dosing regiments. Thedevices may be employed to deliver conjugates and/or particles of thepresent invention across biological tissue, intradermal, subcutaneously,or intramuscularly.

It will be appreciated that the following examples are intended toillustrate but not to limit the present invention. Various otherexamples and modifications of the foregoing description and exampleswill be apparent to a person skilled in the art after reading thedisclosure without departing from the spirit and scope of the invention,and it is intended that all such examples or modifications be includedwithin the scope of the appended claims. All publications and patentsreferenced herein are hereby incorporated by reference in theirentirety.

EXAMPLES Example A: HPLC Analytical Methods: Analysis of the Product byC18 Reverse Phase HPLC (Method 1)

HPLC analysis of the compounds described herein was carried out onZorbax Eclipse XDB-C18 reverse phase column (4.6×100 mm, 3.5 μm, AgilentPN: 961967-902) with a mobile phase consisting of water+0.1% TFA(solvent A) and acetonitrile+0.1% TFA (solvent B at a flow rate of the1.5 mL/min and column temperature of 35° C. The injection volume was 10μL and the analyte was detected using UV at 220 and 254 nm. The gradientis shown in Table 5.

TABLE 5 Gradient Time (mins) % A % B 0 95 5 6 5 95 8 5 95 8.01 95 5 1095 5

Example 1: Synthesis of Conjugate 1

To a solution of cabazitaxel (2.00 g, 2.40 mmol) and2-(2-pyridinyldithio)ethanol p-nitrophenyl carbonate (915 mg, 2.60 mmol)in dichloromethane (48 mL) was added DMAP (439 mg, 3.60 mmol). Thesolution was stirred at room temperature overnight, then washed with0.1N HCl (3×20 mL), saturated aqueous NaCl (50 mL), and dried withsodium sulfate. The solvent was removed in vacuo, the remaining residuepurified by silica gel chromatography (2:1 petroleum ether:ethylacetate) to give cabazitaxel 2-(2-pyridyldithio)ethylcarbonate (2.50 g,2.38 mmol, 99% yield). LCMS m/z: 1049 (M+H).

To a solution of octreotide acetate. (2.08 g, 1.93 mmol) in DMF (20 mL)and diisopropylethylamine (2.0 mL), cooled to −40° C., was added asolution of BocOSu (419 mg, 1.95 mmol) in DMF (5 mL) dropwise. Thereaction was gradually warmed to room temperature, over 3 hours. Most ofthe DMF was removed, and the reaction mixture loaded onto a C18 column,eluting with 15% to 60% acetonitrile in water with 0.1% AcOH, to givethe product as the acetate salt (1.53 g, 1.30 mmol, 67% yield). LCMSm/z: 510.3 (M-Boc+2H)/2.

To a solution of Lys-Boc octreotide acetate (545 mg, 0.462 mmol) in DMF(10 mL) and diisopropylethylamine (1 mL) was added a solution of3-tritylmercaptopropionic acid NHS ester (308 mg, 0.676 mmol) in DMF (4mL). The reaction was stirred at 50° C. for 2 hours, after which HPLCshows complete consumption of starting material. All solvent was removedin vacuo, and the remaining material purified by reverse phasechromatography to give the product (623 mg, 0.430 mmol, 93% yield).

A vial was charged with Lys-Boc octreotide 3-tritylmercaptopropionamide(443 mg, 0.306 mmol), and water (0.25 mL), trifluoroacetic acid (10 mL)and triisopropylsilane (0.25 mL) were added. The reaction was stirred atroom temperature for 10 min, and all solvents were removed in vacuo. Theremaining residue was dissolved in DMF (7 mL) and diisopropylethylamine(0.50 mL). A solution of cabazitaxel 2-(2-pyridyldithio)ethylcarbonate(407 mg, 0.388 mmol) in DMF (3 mL) was added to the solution, and thereaction stirred at room temperature for 1 hour. The reaction was loadedonto a C18 column, eluting with 30% to 70% acetonitrile in water with0.1% AcOH to give the desired product, conjugate 1, as the acetate salt(288 mg, 0.137 mmol, 45% yield). LCMS m/z: 1023.0 (M+2H)/2.

Example 2: Synthesis of Conjugate 2

Octreotide acetate (515 mg, 0.477 mmol) was dissolved in DMF (6 mL) anddiisopropylethylamine (1.0 mL). The solution was cooled to −40° C., anda solution of FMocOSu (182 mg, 0.539 mmol) in DMF (4 mL) was addeddropwise. The reaction was gradually warmed to room temperature over 2hours. pH 8.0 phosphate buffer (1 mL) was added, and the reactionmixture loaded onto a 50 g C18 column. Eluting with 15% to 85%acetonitrile in water gave Lys-Fmoc octreotide (419 mg, 0.337 mmol, 71%yield). LCMS m/z: 621.3 (M+2H)/2.

A flask was charged with doxorubicin (1.39 g, 2.40 mmol) and FMocOSu(1.69 g, 5.00 mmol). DMF (10 mL) and diisopropylethylamine (875 μL, 5.00mmol) were added, and the reaction stirred at room temperature for 3hours. All solvent was removed in vacuo, and the remaining residueloaded on an 80 g silica gel column, eluting with 0% to 8% methanol indichloromethane to give FMoc doxorubicin (1.84 g, 2.40 mmol, 100%yield). LCMS m/z: 397.1 (FMoc daunosamine), 352.2 (M-daunosamine).

A flask was charged with Fmoc doxorubicin (1.84 g, 2.40 mmol) andglutaric anhydride (1.09 g, 9.60 mmol). DMF (10 mL) anddiisopropylethylamine (875 μL, 5.00 mmol) were added, and the reactionstirred at room temperature for 3 hours. Most of the solvent was removedin vacuo, until the total volume was ˜5 mL. This solution was addeddropwise into rapidly stirring 0.1% aqueous trifluoroacetic acid (100mL), cooled to 0° C. The remaining suspension was filtered, theremaining solid washed with water (20 mL), and the solid dried in vacuo.The solid was taken up in 2% methanol in dichloromethane, and loadedonto an 80 g silica gel column. Eluting with 0% to 15% methanol in99.5/0.5 dichloromethane/diisopropylethylamine gave Fmoc doxorubicinhemiglutarate (1.10 g, 1.25 mmol, 52% yield). LCMS m/z: 493.1(M-daunosamine), 397.1 (FMoc daunosamine).

A vial was charged with Fmoc doxorubicin hemiglutarate (104 mg, 0.103mmol), and to this was added a solution of Lys-FMoc octreotide (134mmol, 0.107 mmol) in DMF (3 mL), followed by a solution of TBTU (66.1mg, 0.206 mmol) in DMF (3 mL). Diisopropylethylamine (50 μL, 0.287 mmol)was added, and the reaction stirred at room temperature for 2 h. Allsolvent was removed in vacuo, and the remaining material loaded on a 24g silica gel column. Eluting with 0% to 15% methanol in dichloromethanegave Lys-Fmoc octreotide hemiglutarate FMoc doxorubicin (208 mg, 0.0989mmol, 96% yield).

Lys-Fmoc octreotide hemiglutarate Fmoc doxorubicin (208 mg, 0.0989 mmol)was dissolved in 5 mL DMF, and 1 mL piperidine. After stirring for 30minutes, all solvent was removed in vacuo, and the remaining residue wasdissolved in DMF (1 mL), and this solution added dropwise into rapidlystirring ethyl acetate (100 mL). This suspension was stirred at roomtemperature for 5 min, filtered, the remaining solid washed with ethylacetate (20 mL), and dried in vacuo. The remaining solid was purified byreverse phase chromatography (5% to 50% acetonitrile in water, with 0.1%TFA) to give the desired product as the bis-TFA salt (55.8 mg, 0.0296mmol, 28% yield). LCMS m/z: 829.9 (M+2H)/2.

Example 3: Synthesis of Conjugate 3

To a solution of octreotide acetate (540 mg, 0.501 mmol) in DMF (8 mL)and N,N-diisopropylethylamine (175 μL, 1.00 mmol), cooled to 0° C., wasadded a solution of di-tert-butyl dicarbonate (109 mg, 0.499 mmol) inDMF (7 mL). The reaction was stirred at 0° C. for 1 hour, then at roomtemperature for 1 hour. S-trityl-3-mercaptopropionic acidN-hydroxysuccinimide ester (668 mg, 1.50 mmol) was then added as asolid, and the reaction stirred at room temperature for 16 hours. Thesolvents were removed in vacuo, and the remaining material purified bysilica gel chromatography (0% to 8% methanol in dichloromethane) to giveA (560 mg, 0.386 mmol, 77% yield).

To a solution of 2,2′-dipyridyl disulfide (1.51 g, 6.85 mmol) inmethanol (20 mL) was added 2-(butylamino)ethanethiol (500 μL, 3.38mmol). The reaction was stirred at room temperature for 18 hours, thenthe solvents removed in vacuo. The remaining material was purified bysilica gel chromatography to give disulfide B (189 mg, 0.780 mmol, 23%yield) which was stored at −18° C. until use.

To a solution of cabazitaxel (410 mg, 0.490 mmol) in dichloromethane (10mL) and pyridine (0.50 mL), cooled to −40° C., was added a solution ofp-nitrophenyl chloroformate (600 mg, 2.98 mmol) in dichloromethane (10mL). The reaction was stirred at −40° C. for 2 hours, and the reactionwarmed to room temperature and washed with 0.1N HCl (20 mL). The aqueouslayer was extracted with dichloromethane (2×20 mL), and the combinedorganic layers dried with MgSO₄, and the solvent removed in vacuo. Theremaining material was purified by silica gel chromatography to givecabazitaxel-2′-p-nitrophenylcarbonate (390 mg, 0.390 mmol, 80% yield.)

A solution of cabazitaxel-2′-p-nitrophenylcarbonate (390 mg, 0.390 mmol)in dichloromethane (15 mL) was added to B (190 mg, 0.784 mmol).N,N-diisopropylethylamine (1.0 mL, 5.74 mmol) was added, and thereaction stirred at 30° C. for 18 hours, then the solvents removed invacuo and the remaining material purified by silica gel chromatographyto give the cabazitaxel disulfide (326 mg, 0.295 mmol, 78% yield). ESIMS: calc'd 1103.4. found 1103.9 [M+1].

A vial was charged with A (10.0 mg, 0.00690 mmol), and water (25 μL),trifluoroacetic acid (500 μL) and triisopropylsilane (10 μL) were added.The reaction was stirred at room temperature for 5 min until it turnedcolorless, then all solvents were removed in vacuo. To this residue wasadded the cabazitaxel disulfide (10.4 mg, 0.00942 mmol), pH 8.0phosphate buffer (1.0 mL) and THF (1.0 mL). The reaction was stirred atroom temperature for 2 hours. DMSO (1.0 mL) was added to solubilize anyremaining solid residue, and the resulting solution purified bypreparative HPLC (30% to 85% acetonitrile in water with 0.2% aceticacid) to give the product as the acetate salt (10.3 mg, 0.00477 mmol,69% yield). ESI MS: calc'd 2098.9. found 1050.6 [(M+2)/2].

Example 4: Synthesis of Conjugate 4

A vial was charged with trityl thio octreotide derivative (20.9 mg,0.0144 mmol), and water (25 μL), trifluoroacetic acid (1.0 mL), andtriisopropylsilane (10 μL) were added. The reaction was stirred at roomtemperature until it turned colorless (5 min), then all solvents wereremoved in vacuo. To this residue was added 2,2′-dipyridyl disulfide(10.5 mg, 0.0477 mmol), water (1.0 mL) and methanol (1.0 mL). Thereaction was stirred at room temperature for 2 hours, then DMSO (1.0 mL)was added to solubilize any remaining solids. The reaction was purifiedby preparative HPLC (5% to 50% acetonitrile in water with 0.2% aceticacid) to give the disulfide as the acetate salt (12.6 mg, 0.00987 mmol,69% yield). ESI MS: calc'd 1215.4. found 608.8 [(M+2)/2].

A vial was charged with the disulfide (10.0 mg, 0.00783 mmol) and DM-1(6.00 mg, 0.00813). Phosphate buffer (pH 8, 2.0 mL) and methanol (3.0mL) were added, and the reaction stirred for 2 hours at roomtemperature. DMSO (3.0 mL) was added to solubilize any remaining solidsin the reaction mixture, and the reaction solution purified bypreparative HPLC (25% to 75% acetonitrile in water with 0.2% aceticacid) to give the product as the acetate salt (9.32 mg, 0.00490 mmol,63% yield). ESI MS: calc'd 1841.7, found 912.9 [(M+2-H₂O)/2].

Example 5: Synthesis of Conjugate 5

A vial was charged with trityl thio octreotide derivative (5.2 mg,0.0036 mmol), and water (25 μL), trifluoroacetic acid (500 μL), andtriisopropylsilane (10 μL) were added. The reaction was stirred until itturned colorless (5 minutes), then all solvents were removed in vacuo.To this was added a solution of MC-Val-Cit-PABC-MMAE (4.8 mg, 0.0036mmol) in DMF (1.0 mL). Saturated sodium bicarbonate (100 μL) was added,and the reaction stirred at room temperature for 2 hours. Additionalwater (1 mL) was added, and the resulting solution was purified bypreparative HPLC (30% to 75% acetonitrile in water with 0.2% aceticacid) to yield the product as the acetate salt (4.9 mg, 0.0020 mmol, 56%yield). ESI MS: calc'd 2422.2. found 1212.9 [(M+2)/2].

Example 6: Synthesis of Conjugate 6

A vial was charged with amino-PEG8-acid (221 mg, 0.501 mmol) and trityl3-mercaptopropionic acid NHS ester (223 mg, 0.501 mmol). DMF (5 mL) anddiisopropylethylamine (500 μL) were added, and the reaction stirred atroom temperature for 24 hours. After 24 hours, DCC (206 mg, 1.00 mmol)and N-hydroxysuccinimide (115 mg, 1.00 mmol) were added, and thereaction stirred for another 24 hours. The reaction mixture wasfiltered, washing the solid with 3 mL DMF, and the collected filtratewas concentrated in vacuo. The remaining material was purified by silicagel chromatography to give the product (406 mg, 0.467 mmol, 93% yield).

Octreotide acetate (335 mg, 0.311 mmol) was dissolved in DMF (5 mL), thesolution cooled to 0° C., diisopropylethylamine (150 μL) was added, anda solution of Boc₂O (67.9 mg, 0.311 mmol) in DMF (3 mL) was addeddropwise. The reaction was stirred at 0° C. for 30 minutes, then warmedto room temperature for 30 minutes. A solution of the NHS ester in 5 mLDMF was then added, and the reaction stirred at room temperature for 3days. All solvent was removed in vacuo, and the remaining residuepurified by silica gel chromatography to give the product (249 mg, 0.133mmol, 43% yield).

The starting material (21.2 mg, 0.0113 mmol) was dissolved in TFA (1mL), water (25 μL), and triisopropylsilane (25 μL). The reaction wasstirred at room temperature for 5 min, and all solvent was removed invacuo. The remaining residue was dissolved in DMF (1 mL), and a solutionof 2,2′-dipyridyldisulfide (15.0 mg, 0.0681 mmol) in DMF (1 mL) wasadded, followed by diisopropylethylamine (200 μL). The reaction wasstirred at room temperature for 10 minutes, and purified by preparativeHPLC. The intermediate 2-pyridyldisulfide was dissolved in DMF (1 mL),and a solution of DM-1 (6.0 mg, 0.0081 mmol) in DMF (1 mL) was added,followed by diisopropylethylamine (200 μL). The reaction was stirred atroom temperature for 15 minutes, and the reaction mixture was purifiedby preparative HPLC to give the product (10.1 mg, 0.00445 mmol, 39%yield).

Example 7: Synthesis of Conjugate 7

A mixture of Lys-Boc octreotide free base (50.0 mg, 0.0447 mmol),(Boc)HNCys(Trt)-[Lys(Boc)]₄—OH (80.0 mg, 0.0581 mmol), and EDC (19.1 mg,0.100 mmol) in dichloromethane (3.0 mL) and diisopropylethylamine (0.20mL) was stirred for 24 hours. The reaction was loaded onto a silica gelcolumn, and eluting with 0% to 15% methanol in dichloromethane gaveBocHN-Cys(Trt)-[Lys(Boc)]4-octreotide(Lys-Boc) (24.0 mg, 0.00968 mmol,22% yield).

BocHN-Cys(Trt)-[Lys(Boc)]4-octreotide(Lys-Boc) (24.0 mg, 0.00968 mmol)was dissolved in water (25 μL), TFA (1 mL), and triisopropylsilane (25μL). The reaction was stirred at room temperature for 5 minutes, and allsolvent was removed in vacuo. The remaining residue was dissolved in DMF(1 mL), and a solution of 2,2′-dipyridyldisulfide (15.0 mg, 0.0681 mmol)in DMF (1 mL) was added, followed by diisopropylethylamine (100 μL). Thereaction was stirred at room temperature for 5 minutes, and purified bypreparative HPLC. The isolated pyridyl disulfide was dissolved in DMF (1mL), and a solution of DM-1 (5.2 mg, 0.070 mmol) in DMF (1 mL) wasadded, followed by diisopropylethylamine (100 μL). The reaction wasstirred at room temperature for 10 minutes, then purified by preparativeHPLC to give the product (3.0 mg, 0.0013 mmol, 13% yield).

Example 8: Inhibition of Cell Proliferation by Conjugates

Conjugates were assessed in an in vitro assay evaluating inhibition ofcell proliferation. NCI-H524 (ATCC) human lung cancer cells were platedin 96 well, V-bottomed plates (Costar) at a concentration of 5,000cells/well and 24 hours later were treated with compound for 2 hours andfurther incubated 70 hours. Compound starting dose was 20 μM andthree-fold serial dilutions were done for a total of ten points. After 2hours of treatment, cells were spun down, the drug containing media wasremoved, and fresh complete medium was added and used to resuspend thecells, which were spun again. After removal of the wash media, the cellswere resuspended in complete medium, then transferred into white walled,flat bottomed 96 well plates. Cells were further incubated for anadditional 70 hours to measure inhibition of cell proliferation.Octreotide alone had no significant effect on cell proliferation.Proliferation was measured using CellTiter Glo reagent using thestandard protocol (Promega) and a Glomax multi+detection system(Promega). Percent proliferation inhibition was calculated using thefollowing formula: % inhibition=(control-treatment)/control*100. Controlis defined as vehicle alone. IC₅₀ curves were generated using thenonlinear regression analysis (four parameter) with GraphPad Prism 6.Data for representative compounds (Conjugates 1-7) were shown in Table6. IC₅₀ values for representative compounds with octreotide competitionwere also measured and were shown in Table 6.

TABLE 6 IC₅₀ of conjugates 1-7 H524 IC₅₀ ₊ 100 μM Conjugate H524 IC₅₀(nM) octreotide (nM) 1 110 146 2 2850 2940 3 >20000 >20000 4 229 853 5433 826 6 234 629 7 955 1770

These data demonstrate that conjugates retain the ability to bind tosomatostatin and internalize the receptor. In some instances, this alsoshows that the linker is cleaved to activate the cytotoxic payloadeffectively to kill the tumor cells.

Example 9: Ki of Conjugates for Somatostatin Receptor

Two conjugates were assessed in an in vitro assay evaluating binding tothe somatostatin receptor 2 (SSTR2). A radioligand-receptor bindingassay was conducted at Eurofins Panlabs (Taiwan) to determine theaffinity of conjugates described herein to the SSTR2. The assay measuresbinding of radiolabeled ligand, [¹²⁵ I] labeled somatostatin, to humanSSTR2 using membrane preparations from SSTR2 expressing CHO-K1 cells.Membranes were incubated with radiolabeled somatostatin (0.03 nM) in thepresence of conjugate/compound starting at a dose of 10 uM using 6×serial dilutions to obtain a 10-pt curve. After a four-hour incubation,membranes were filtered and washed 3× and counted to determine theremaining [¹²⁵ I] somatostatin bound to the receptor. IC50 values weredetermined by a non-linear, least squares regression analysis usingMathIQ™ (ID Business Solutions Ltd., UK). The Ki values were calculatedusing the equation of Cheng and Prusoff (Cheng and Prusoff, Biochem.Pharmacol. 22:3099-3108, 1973) using the observed IC50 of the testedconjugate/compound, the concentration of radioligand employed in theassay, and the historical values for the KD of the ligand obtained atEurofins.

TABLE 7 Ki of conjugates 1-2 Conjugate SSTR2 Ki (nM)  1 0.800  2 0.24010 0.100 76 0.120 78 0.190

These data demonstrate that the high affinity of the peptide for thereceptor is retained after addition of the linker and drug to thepeptide.

Example 10: Internalization of Conjugates to Somatostatin Receptor

Two conjugates were assessed in an in vitro assay evaluating SSTR2. Thesteps outlined below provide the assay volumes and procedure forperforming agonist assays using the PathHunter eXpress Activated GPCRInternalization cells and PathHunter Detection Reagents generallyaccording to the manufacturer's recommendations. GraphPad Prism® wasused to plot the agonist dose response.

TABLE 8 EC₅₀ of conjugates 1-2 Conjugate SSTR2 EC₅₀ (nM) 1 4.4 2 35 763.0

These data demonstrate that the conjugates potently induceinternalization of the receptor as a mechanism for the selectivedelivery of a conjugate to the cytoplasm of SSTR2 expressing cells.

Example 11: Nanoparticle Formulation of Conjugate 1

Nanoparticle formulation of conjugate 1. Octreotide-cabazitaxelconjugate 1 was successfully encapsulated in polymeric nanoparticlesusing a single oil in water emulsion method (refer to Table 9A and Table9B below). In a typical water-emulsion method, the drug and a suitablepolymer or block copolymer or a mixture of polymers/block copolymers,were dissolved in organic solvents such as dichloromethane (DCM), ethylacetate (EtAc) or chloroform to form the oil phase. Co-solvents such asdimethyl formamide (DMF) or acetonitrile (ACN) or dimethyl sulfoxide(DMSO) or benzyl alcohol (BA) were sometimes used to control the size ofthe nanoparticles and/or to solubilize the drugs. A range of polymersincluding PLA97-b-PEG5, PLA35-b-PEG5 and PLA16-b-PEG5 copolymers wereused in the formulations. Nanoparticle formulations were prepared byvarying the lipophilicity of conjugate 1. The lipophilicity was variedby using hydrophobic ion-pairs of conjugate 1 with differentcounterions. Surfactants such as Tween® 80, sodium cholate, Solutol® HSor phospholipids were used in the aqueous phase to assist in theformation of the fine emulsion. The oil phase was slowly added to thecontinuously stirred aqueous phase containing an emulsifier (such asTween® 80) at a typical 10%/90% v/v oil/water ratio and a coarseemulsion was prepared using a rotor-stator homogenizer or an ultrasoundbath. The coarse emulsion was then processed through a high-pressurehomogenizer (operated at 10,000 psi) for N=4 passes to form ananoemulsion. The nanoemulsion was then quenched by a 10-fold dilutionwith cold (0-5° C.) water for injection quality water to remove themajor portion of the ethyl acetate solvent resulting in hardening of theemulsion droplets and formation of a nanoparticle suspension. In somecases, volatile organic solvents such as dichloromethane can be removedby rotary evaporation. Tangential flow filtration (500 kDa MWCO, mPESmembrane) was used to concentrate and wash the nanoparticle suspensionwith water for injection quality water (with or withoutsurfactants/salts). A cryoprotectant serving also as tonicity agent(e.g., 10% sucrose) was added to the nanoparticle suspension and theformulation was sterile filtered through a 0.22 μm filter. Theformulation was stored frozen at ≤−20° C. Particle size (Z-ave) and thepolydispersity index (PDI) determined by dynamic light scattering of thenanoparticles were characterized by dynamic light scattering, assummarized in the table below. The actual drug load was determined usingHPLC and UV-visible absorbance. This was accomplished by evaporating thewater from a known volume of the nanoparticle solution and dissolvingthe solids in an appropriate solvent such as DMF. The drug concentrationwas normalized to the total solids recovered after evaporation.Encapsulation efficiency was calculated as the ratio between the actualand theoretical drug load.

Formulations Using Free Conjugate 1.

Conjugate 1 was observed to have a high solubility in aqueous mediacontaining surfactants such as Tween® 80 and forms mixed micelles. Incertain formulations, conjugate 1 was used without any changes to itsnative lipophilicity (free conjugate). Surprisingly, even with a highsolubility of conjugate 1 in aqueous Tween® 80, the free conjugateexhibited a high degree of encapsulation in the nanoparticles. Withoutcommitting to any particular theory, the tendency of conjugate 1 toretain in the nanoparticle despite a high aqueous solubility inTween®/water could be due to the high lipophilicity of cabazitaxel andits compatibility/miscibility with the polymeric matrix. The presence oftwo phenylalanine amino acids in the octreotide peptide may also assistin the interaction of the conjugate with the polymeric matrix.

Formulations Using Hydrophobic Ion Pairing (HIP) of Conjugate 1

HIP techniques were used to enhance the lipophilicity of conjugate 1.The conjugate has one positively charged moiety, on the lysine aminoacid. A negatively charged dioctyl sodium sulfosuccinate (AOT) moleculeswas used for everyone molecule of the conjugate to form the HIP. Theconjugate and the AOT were added to a methanol, dichloromethane andwater mixture and allowed to shake for 1 hour. After further addition ofdichloromethane and water to this mixture, the conjugate 1/AOT HIP wasextracted from the dichloromethane phase and dried. Sometimes, DMF wasused to solubilize the HIP complex. The results of the formulations aresummarized in Table 9A and 9B.

TABLE 9A Formulations of conjugate 1 nanoparticles using free drugconjugate (DC) Formulation # NP3 NP4 NP6 Process Single Single SingleSingle Single Single Single emulsion emulsion emulsion emulsion emulsionemulsion emulsion Polymer PLA97- PLA16- PLA16- PLA97- PLA16- PLA16-PLA35- mPEG5 mPEG5 mPEG5 mPEG5 mPEG5 mPEG5 mPEG5 Polymer 100 100 100 100100 100 concentration, mg/mL Emulsion 20 20 20 20 20 20 20 Volume, mLOil phase 10% DMF/ 10% DMF/ 10% DMF/ 10% DMF/ 10% DMF/ 10% DMF/ 20% DMF/90% DCM 90% DCM 90% DCM 90% EA 90% EA 90% EA 80% EtOAc Aqueous cold coldcold cold cold cold cold phase 0.3% 0.3% water/EA 0.2% water/EA 0.1%0.1% DiOctPC DiOctPC Tween ® Tween ® DiOctPC in water in water 80 in80/EA in water/ water/EA EtOAc Oil phase 10.00% 10.00% 10.00% 10.00%10.00% 10.00% 10.00% volume fraction, % Wash* ×10 cold ×10 cold ×20 coldTween ® Tween ® Tween ® Tween ® water water water 80 (0.5%) 80 (0.2%) 80(0.2%) 80 (0.2%) and ×30 and ×25 and ×25 and ×25 cold cold cold coldwater water water water Z.ave/PDI 163.1/0.11 69.3/0.159 49.2/0.265106.8/ 95.6/0.564 44.95/ 69.4/0.190 (quenched one one 0.192 one 0.191Emulsion) peak peak peak Z.ave/PDI 176(0.214) 78 50 (0.6) 101.185.2/0.632 44.8/ 62.7/0.145 (post TFF bump (0.278) bimodal (0.194) 0.127filtered) at small bump at distribution one peak sizes small with peakssizes at ~150 and 13 mn TDL (wt %) 9.27 9.18 9.26 9.13 9.31 5.93 4.87ADL (wt %) 8.13 8.76 8.64 8.26 8.64 5.49 4.76 EE = 87.7 95.4 93.4 96.8592.73% 92.49% 102.32% ADL/TDL, % Potency, 0.52 0.54 0.49 0.45 0.8350.228 0.52 mg/mL

TABLE 9B Formulations of conjugate 1 nanoparticles using conjugate 1/AOTHIP Formulation # NP1 NP2 Process Single emulsion Single emulsion Singleemulsion Single emulsion Polymer PLA16-mPEG5 PLA97-mPEG5 PLA35-mPEG5PLA16-mPEG5 Polymer 100 100 100 100 concentration, mg/mL Emulsion 20 2020 20 Volume, mL Oil phase 10% DMF/90% DCM 20% DMF/80% EtOAc 20% DMF/80%EtOAc 10% DMF/90% EtOAc Aqueous cold water cold 0.2% DiOctPC in cold0.2% DiOctPC in cold 0.2% DiOctPC in phase water/EtOAc water/EtOAcwater/EtOAc Oil phase 10.00% 10.00% 10.00% 10.00% volume fraction, %Wash* Tween ® 80 (0.2%) Tween ® 80 (0.2%) Tween ® 80 (0.2%) Tween ® 80(0.2%) and saline ×15 cold and saline ×25 cold and saline ×20 cold andsaline ×20 cold water water water water Z.ave/PDI 100/0.26 one major106/0.09 one peak 102/0.05 one peak 86.6/0.123 (quenched peak Emulsion)Z.ave/PDI 90/0.28 one major 91/0.1 one peak 75/0.08 one peak 54/0.184(post TFF peak filtered) TDL (wt %) 4.10 6.50 3.6 5.65 ADL (wt %) 4.896.73 3.62 5.7 EE = 120.0 103.0 100 101 ADL/TDL, % Potency, 0.17 0.660.44 0.503 mg/mL TDL: Theoretical Drug Loading ADL: Actual Drug LoadingNA: not available EE: encapsulation efficiency *Washing was optimizedfor each nanoparticle formulation.

These data demonstrate that somatostatin receptor targeted conjugatescan be efficiently and effectively encapsulated in nanoparticles.

Example 12: Nanoparticles Containing Conjugate 2

Conjugate 2 was successfully encapsulated in polymeric nanoparticlesusing a single oil in water emulsion method (refer to Table 6A and 6Bbelow). In a typical water-emulsion method, the drug and a suitablepolymer or block copolymer or a mixture of polymers/block copolymers,were dissolved in organic solvents such as dichloromethane (DCM), ethylacetate (EtAc) or chloroform to form the oil phase. Co-solvents such asdimethyl formamide (DMF) or acetonitrile (ACN) or dimethyl sulfoxide(DMSO) or benzyl alcohol (BA) were sometimes used to control the size ofthe nanoparticles and/or to solubilize the drugs. A range of polymersincluding PLA97-b-PEG5, PLA74-b-PEG5, PLA35-b-PEG5 and PLA16-b-PEG5copolymers were used in the formulations. Nanoparticle formulations wereprepared by varying the lipophilicity of conjugate 2. The lipophilicityof conjugate 2 was varied by using hydrophobic ion-pairs of conjugate 2with different counterions. Surfactants such as Tween® 80, sodiumcholate, Solutol® HS or lipids were used in the aqueous phase to assistin the formation of the fine emulsion. The oil phase was slowly added tothe continuously stirred aqueous phase containing an emulsifier (such asTween® 80) at a typical 10/90% v/v oil/water ratio and a coarse emulsionwas prepared using a rotor-stator homogenizer or an ultrasound bath. Thecoarse emulsion was then processed through a high-pressure homogenizer(operated at 10,000 psi) for N=4 passes to form a nanoemulsion. Thenanoemulsion was then quenched by a 10-fold dilution with cold (0-5° C.)water for injection quality water to remove the major portion of theethyl acetate solvent resulting in hardening of the emulsion dropletsand formation of a nanoparticle suspension. In some cases, volatileorganic solvents such as dichloromethane can be removed by rotaryevaporation. Tangential flow filtration (500 kDa MWCO, mPES membrane)was used to concentrate and wash the nanoparticle suspension with waterfor injection quality water (with or without surfactants/salts). Alyoprotectant (e.g., 10% sucrose) was added to the nanoparticlesuspension and the formulation was sterile filtered through a 0.22 μmfilter. The formulation was stored frozen at ≤−20° C. Particle size(Z-avg.) and the polydispersity index (PDI) of the nanoparticles werecharacterized by dynamic light scattering, as summarized in the tablebelow. The actual drug load was determined using HPLC and UV-Visabsorbance. This was accomplished by evaporating the water from a knownvolume of the nanoparticle solution and dissolving the solids in anappropriate solvent such as DMF. The drug concentration was normalizedto the total solids recovered after evaporation. Encapsulationefficiency was calculated as the ratio between the actual andtheoretical drug load.

In some formulations, conjugate 2 was used without any changes to itsnative lipophilicity (free conjugate). Surprisingly, even with a highsolubility of 2 in aqueous Tween® 80, and the hydrophilic nature ofoctreotide, the free conjugate exhibited a high degree of encapsulationin the nanoparticles. The tendency of 2 to be retained in thenanoparticle was reduced compared to 1.

Formulations Using HIP of Conjugate 2

Hydrophobic ion-pairing (HIP) techniques were used to enhance thelipophilicity of conjugate 2. The conjugate has two basic moieties, onthe lysine and on the doxorubicin. A negatively charged dioctyl sodiumsulfosuccinate (AOT) molecule was used for every molecule of theconjugate to form the HIP. The conjugate and the AOT were added to amethanol, dichloromethane and water mixture and allowed to shake for 1hour. After further addition of dichloromethane and water to thismixture, the conjugate 2/AOT HIP was extracted from the dichloromethanephase and dried. In some cases, DMF was used to solubilize the HIPcomplex. Specifications and data are shown in Table 10A and Table 10B.

Example of Preparing an HIP Complex of Conjugate 2 with AOT

#Positive charges on conjugate 2=2; MW=1658.9 g/mol

Mass of conjugate 2=34.5 mg.

#moles of conjugate 2=0.0208 mmoles

Moles of AOT required to cover the 2 positive charges=0.0416 mmoles.

Weight of AOT (mg) [MW=445 g/mol]=18.5 mg

Conjugate 2 and AOT were added to a solution of 1 mL of water and 2.1 mLof methanol. 1 mL of dichloromethane was added to this mixture. A clearred homogenous solution was obtained. This solution was shaken for ataround 30 minutes. 1 mL water and 1 mL of dichloromethane were added tothe solution and the mixture was shaken briefly. The two phases wereallowed to separate. Sometimes in order to accelerate the separation ofthe two phases, the mixture may be centrifuged. The bottom phaseconsisted primarily of dichloromethane whereas the top phase (aqueousphase) was predominantly made of water and methanol. After the formationof the conjugate 2:AOT HIP complex, the lipophilicity introduced ontothe compound increased its solubility in the dichloromethane phase. TheHIP complex was then recovered from the bottom phase and thedichloromethane was evaporated. Sometimes additional dichloromethane wasadded to the remaining aqueous phase to extract the remaining conjugate2:AOT HIP complex.

TABLE 10A Formulations of conjugate 2 nanoparticles using free drugconjugate (DC) Formulation # NP2 NP5 Process Single emulsion Singleemulsion Polymer PLA16-mPEG5 PLA35-mPEG5 Polymer concentration, mg/mL 80160 Emulsion Volume, mL 20 20 Oil phase 20% BA/80% EA 20% BA/80% EAAqueous phase cold (ice) 0.15% Tween ® cold 0.1% Tween ® 80 80/EA&BAWater/EA Oil phase volume fraction, % 10% 10% Wash* ×25 with cold waterdiluted ×10 ×25 with cold water and RT water and concentrated 10concentrated fold Z.ave/PDI (quenched 46.3/0.065 92.7/0.20 Emulsion)Z.ave/PDI (post TFF filtered) 44.5/0.054 79.6/0.09 TDL (wt %) 5.88 3.03ADL (wt %) 3.60 1.44 EE = ADL/TDL, % 61.2 47.6 Potency, mg/mL 0.170 0.30

TABLE 10B Formulations of conjugate 2 nanoparticles using conjugate2/AOP HIP Formulation # NP1 NP3 NP4 NP6 NP7 NP8 Process Single SingleSingle Single Single Single emulsion emulsion emulsion emulsion emulsionemulsion Polymer PLA97- PLA35- PLA16- PLA74- PLA35- PLA97- mPEG5 mPEG5mPEG5 mPEG5 mPEG5 mPEG5 Polymer 100 100 100 100 100 100 concentration,mg/mL Emulsion 20 20 20 20 20 20 Volume, mL Oil phase 92% EA/ 83% EA/85% EA/ 80% EA/ 80% EA/ 80% EA/ 8% DMF 17% DMF 15% DMF 20% DMF 20% DMF20% DMF Aqueous cold (ice) cold 0.1% cold 0.1% cold 0.2% cold 0.1% cold(ice) phase 0.2% Tween ® 80 Tween ® Tween ® 80 Tween ® 80 0.2% DiOctPCWater/EA 80 Water/EA Water/EA DiOctPC Water/EA Water/EA Water/EA Oilphase 7.50% 10% 10% 10% 10% 10.00% volume fraction, % Wash* ×25 with ×25with ×25 with ×25 with ×15 with ×15 with cold water, 1XPBS 1XPBS 1XPBSsaline, ×5 cold saline, diluted ×10 diluted ×10 diluted diluted ×10 withwater, ×5 with cold RT water RT water ×10 RT RT water warmed to water,warm and and water and and 37 and to 37° C. for concentratedconcentrated concentrated concentrated diluted ×10 3 min, 10 fold 10fold 10 fold RT water diluted ×10 and RT water concentrated and 10 foldconcentrated Z.ave/PDI 98.4/0.08 70.44/0.106 62.78/0.27  110.6/0.20793.16/0.232   96.5/0.11 (quenched Emulsion) Z.ave/PDI 88.3/0.0566.55/0.073 65.65/0.258 100.7/0.127 80.86/0.153 96.1.3/0.10 (post TFFfiltered) TDL (wt %) 4.2 4.52 8.39 6.89 7.63 7.7 ADL (wt %) 3.7 4.076.85 4.69 7.39 6.4 EE = 87 89.9 81.6 68.1 96.9 83 ADL/TDL, % Potency,0.25 0.4 0.73 0.28 0.47 0.316 mg/mL * Washing was optimized for eachnanoparticle formulation.

These data further demonstrate that somatostatin receptor targetedconjugates can be efficiently and effectively encapsulated innanoparticles.

Example 13: Pharmacokinetics of Nanoparticle Formulations of 1 and 2

Nanoparticles are typically formulated for in vivo delivery in 10%sucrose and free drug formulations varied, but are typically dosed in10% Solutol®/10% sucrose, or physiological saline. In this exampleconjugate 1 without nanoparticle formulation was dosed as a solution in20% propyleneglycol/80% aqueous sucrose (10%).

For rat pharmacokinetic studies using nanoparticles as described herein,a 0.1 mg/mL solution was dosed at 10 mL/kg such that a 1 mg/kg IV bolusdose was introduced by tail vein injection into rats. Following compoundadministration, blood was collected at 0.083 hours, 0.25 hours, 0.5hours, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours post dose intolithium heparin coated vacuum tubes. Tubes were inverted for 5 minutesand then placed on wet ice until centrifuged for 5 minutes at 4° C. at6000 rpm. Plasma was harvested, frozen at −80° C. and shipped to forbioanalysis on dry ice.

50 uL of rat plasma was precipitated with 300 uL of DMF and theresulting supernatant was measured for compound content by LC-MS/MSelectrospray ionization in the positive mode.

Representative dose normalized rat pharmacokinetic curves for conjugate1 and nanoparticle formulations of conjugate 1 are shown in FIG. 1.Table 11 shows the normalized area under the curve (AUC) calculationsfor conjugate 1 and the nanoparticles comprising conjugate 1 in FIG. 1.

TABLE 11 AUC of conjugate 1 and nanoparticle formulations 1 NP1 NP2 NP4NP6 AUC (0-inf) 18.3 42.5 154 127 256 μmol/l*h

Representative dose normalized rat pharmacokinetic curves for conjugate2 and nanoparticle formulations of conjugate 2 are shown in FIG. 2.Table 12 shows the normalized area under the curve (AUC) calculationsfor conjugate 2 and the nanoparticles comprising conjugate 2 in FIG. 2.

TABLE 12 AUC of conjugate 2 and nanoparticle formulations 2 NP1 NP3 NP5AUC (0-inf) 14.3 16.0 21.8 29.5 μmol/l*h

These data demonstrate that nanoparticles increase the AUC ofconjugates, thereby demonstrating that targeted nanoparticles can besynthesized using methods described herein having desirable propertiesindicative of improved use for drug delivery, for example, delivery of achemotherapeutic agent to a tumor.

Example 14: Synthesis of DM1 Conjugates

Conjugates comprising DM1 were synthesized according to the followingprocedures:

Synthesis of Intermediates

Nα-Me-Nα-Fmoc-Nε-Boc-lysine (640 mg, 1.31 mmol) was dissolved in dioxane(5 mL) and 4N HCl (5 mL). The reaction was stirred at room temperatureuntil LCMS shows complete deprotection. The reaction mixture waspurified by reverse phase chromatography to give Na-Me-Na-Fmoc-lysine(2′, 500 mg, 1.31 mmol, 100% yield). This material was dissolved in DMF(5 mL) and diisopropylethylamine (0.50 mL), and trityl3-mercaptopropionic acid NHS ester (875 mg, 1.98 mmol) was added. Thereaction was stirred at room temperature, and purified by reverse phasechromatography to give Nα-Me-Nα-Fmoc-Nε-(STrt-propionate)-lysine (3′,600 mg, 0.843 mmol, 64% yield).

Fmoc-threonine(tBu)-OH was loaded onto 2-chlorotrityl resin (3.0 gresin, 1.5 mmol/g loading). Iterative deprotection with 4:1DMF:piperidine, and coupling subsequently with Nα-Fmoc-Nε-Boc-lysine,Nα-Fmoc-N^(in)-Boc-D-tryptophan, Fmoc-tyrosine(tBu),Nα-Me-Nα-Fmoc-Nε-(3STrt-propionate)-lysine, and Fmoc-phenylalanine usingstandard SPPS conditions gave the linear peptide bound to the resin.Resin cleavage with 1% TFA in dichloromethane, followed by cyclizationby dropwise addition of a solution of the linear peptide in 10 mL DMF toa flask with HATU (1.71 g, 4.5 mmol) and HOAt (0.6 M solution, 7.5 mL,4.5 mmol) in DMF (45 mL) and diisopropylethylamine (3.0 mL). Afterstirring for 3 h at room temperature, all DMF was removed in vacuo, andthe remaining material treated with 95:2.5:2.5 TFA:EDT:water for 30 min,the solvent removed in vacuo, and the remaining material purified byreverse phase chromatography to providecyclo[Phe-Nα-Me-Nε-(3STrt-propionate)-Lys-Tyr-DTrp-Lys-Thr] (4′, 427 mg,0.447 mmol, 10% overall yield). LCMS M/Z: 956.5 [M+1].

Compound 5′ was made in an analogous manner to compound 4′.

To a solution of 5′ trifluoroacetate salt (380 mg, 0.387 mmol) in DMF (5mL) and diisopropylethylamine (0.50 mL) was added a solution of BocOSu(91.5 mg, 0.425 mmol) in DMF (2 mL). The reaction was stirred at roomtemperature for 4 h, then the reaction mixture loaded onto a 50 g C18Isco column. Eluting with 5% to 75% acetonitrile in water with 0.1% AcOHyielded 6′ (338 mg, 0.349 mmol, 90% yield).

A vial was charged with cyclo[Phe-NMeGlu-Tyr-DTrp-Lys(Boc)-Thr] (6′,41.8 mg, 0.0431 mmol), 3-maleimidopropylamine HCl (32.9 mg, 0.172 mmol)and COMU (73.7 mg, 0.172 mmol). DMF (1 mL) and diisopropylethylamine(0.10 mL) were added, and the reaction stirred at room temperature for30 min. After 30 min, additional 3-maleimidopropylamine HCl (32.9 mg,0.172 mmol) and diisopropylethylamine (0.10 mL) were added, and thereaction stirred for another 3 h. The reaction was acidified by additionof acetic acid (0.30 mL), water (1 mL) was added to solubilize anymaterial that had come out of solution. The reaction mixture waspurified by preparative HPLC (5% to 75% acetonitrile in water with 0.1%AcOH) to give 7′ (25.7 mg, 0.0233 mmol, 54% yield).

A vial was charged with cyclo[Phe-NMeGlu-Tyr-DTrp-Lys(Boc)-Thr] (6′,39.0 mg, 0.0402 mmol), S-Trt cystamine (38.6 mg, 0.121 mmol) and TBTU(38.8 mg, 0.121 mmol). DMF (1.0 mL) and diisopropylethylamine (0.10 mL)were added, and the reaction stirred at room temperature for 2 h. Thereaction mixture was purified by preparative. HPLC (5% to 95%acetonitrile in water with 0.1% AcOH) to give 8′ (36.2 mg, 0.0285 mmol,71% yield).

A vial was charged with Fmoc-STrt-cysteine (585 mg, 1.00 mmol) and TBTU(330 mg, 1.03 mmol). DMF (4 mL), tert-octylamine (0.176 mL, 1.10 mL) anddiisopropylethylamine (0.30 mL) were added, and the reaction stirred atroom temperature for 16 h. Piperidine (2 mL) was added, the reactionstirred at room temperature for 30 min, and the reaction mixture loadedonto a 50 g C18 Isco column, eluting with 15% to 85% acetonitrile inwater with 0.1% AcOH. The purified product was dried-in vacuo, and theisolated product was redissolved in methanol (10 mL). 1N HCl (2 mL) wasadded, and all solvents removed in vacuo again to give S-trityl cysteinetert-octyl amide HCl salt (401 mg, 0.784 mmol, 78% yield).

A vial was charged with cyclo[Phe-NMeGlu-Tyr-DTrp-Lys(Boc)-Thr] (6′,27.0 mg, 0.0279 mmol), S-trityl cysteine tert-octyl amide HCl (20.0 mg,0.0391 mmol) and TBTU (11.0 mg, 0.0343 mmol). DMF (1 mL) anddiisopropylethylamine (0.10 mL) were added, and the reaction stirred atroom temperature for 1 h. The reaction mixture was purified bypreparative HPLC (25% to 95% acetonitrile in water with 0.1% AcOH) togive 9′ (24.2 mg, 0.0170 mmol, 61% yield).

A flask was charged with trityl-3-mercaptopropionic acid NHS ester (1.02g, 2.29 mmol), and this dissolved in DMF (10 mL). The reaction wascooled to 0° C., and 4,7,10-trioxa-1,13-tridecanediamine (3.00 mL, 13.7mmol) was added all at once. The reaction was stirred at 0° C. for 10min, then warmed to room temperature and stirred for 2 h. The reactionmixture was loaded onto a 100 C18 Isco column, and eluting with 5% to85% acetonitrile in water yielded 10′ (552 mg, 1.00 mmol, 44% yield).

Compounds 11′-13′ were made in an analogous manner to 10′:

A vial was charged with 6′ (27.6 mg, 0.0285 mmol), 10′ (31.4 mg, 0.0570mmol) and TBTU (18.3 mg, 0.0570 mmol). DMF (1 mL) anddiisopropylethylamine (0.10 mL) were added, and the reaction stirred atroom temperature for 2 h. The reaction mixture was purified by prep HPLC(25% to 95% acetonitrile in water with 0.1% AcOH) to give 14′ (29.6 mg,0.0197 mmol, 69% yield).

Compounds 15′-17′ were made in an analogous manner to 14′:

A vial was charged with N-Boc-glutamic acid (125 mg, 0.506 mmol),3-maleimidopropylamine HCl (200 mg, 1.05 mmol) and TBTU (330 mg, 1.03mmol), DMF (5 mL) and diisopropylethylamine (0.30 mL) were added, themixture sonicated for 10 min to give a smooth suspension, and thereaction stirred at room temperature for 18 h. The reaction wasacidified with acetic acid (0.50 mL), and then diluted with water (2.0mL). The reaction mixture was loaded onto a 30 g C18 Isco column, andeluting with 5% to 60% acetonitrile in water with 0.1% AcOH gave 18′(77.0 mg, 0.148 mmol, 29% yield). LCMS M/Z: 520.2 (M+1).

Compound 19′ was made in an analogous manner:

A vial was charged with 18′ (12.0 mg, 0.0233 mmol), and TFA (1 mL) wasadded. The reaction stirred at room temperature for 5 min, then all TFAwas removed in vacuo. In a second vial,cyclo[Phe-NMeGlu-Tyr-DTrp-Lys(Boc)-Thr] (6′, 20.0 mg, 0.0206 mmol) andTBTU (9.0 mg, 0.0280 mmol) were dissolved in DMF (1 mL), anddiisopropylethylamine (0.15 mL). This solution was stirred at roomtemperature for 5 min, then added to the vial with deprotected 18′. Thereaction was stirred at room temperature for 2 h, and the reaction wasacidified by adding AcOH (0.20 mL). The reaction was then purified byprep HPLC, eluting with 15% to 80% acetonitrile in water with 0.2% AcOHto give 20′ (11.6 mg, 0.0085 mmol, 41% yield).

Compound 21′ was made in an analogous manner to 20′:

A vial was charged with 2,2′-dithiodipyridine (110 mg, 0.500 mmol) andthis dissolved in methanol (1 mL). A solution of2-(butylamino)ethanethiol (37.0 μL, 0.250 mmol) in methanol (1 mL) wasadded dropwise, stirred for 5 min, and all methanol removed in vacuo. Tothe remaining residue was added a solution ofcyclo[Phe-NMeGlu-Tyr-DTrp-Lys(Boc)-Thr] (6′, 28.6 mg, 0.0295 mmol) andTBTU (14.2 mg, 0.0443 mmol) in DMF (2 mL) and diisopropylethylamine(0.10 mL). The reaction was stirred at room temperature for 1 h, and thereaction mixture was then loaded onto a 30 g C18 Isco column, elutingwith 40% to 95% acetonitrile in water with 0.1% AcOH to give 22′ (21.1mg, 0.0177 mmol, 60% yield). LCMS M/Z: 547.4 [(M+2-Boc)/2].

Compound 23′ was made in an analogous manner to 22′:

f-cyclo(CTwKTC)T-OH (24′) was synthesized by loading Fmoc-threonine(tBu)onto 2-chlorotrityl resin, and by appending the subsequent amino acidsby standard Fmoc chemistry, with Fmoc-S-trityl-cysteine,Fmoc-threonine(tBu), Nα-Fmoc-Nε-Boc-lysine,Nα-Fmoc-N^(in)-Boc-D-tryptophan, Fmoc-tyrosine(tBu),Fmoc-S-trityl-cysteine, and Fmoc-D-phenylalanine. Final deprotection wasachieved by treating with 95:2.5:2.5 TFA:water:triisopropylsilane. Thecrude peptide was dried, weighed, dissolved in 1:1 acetonitrile:water,and treated with 2 equiv. iodine in methanol to give the cyclicdisulfide. Purification on reverse phase gave the desired peptide.

Compounds 25′-28′ were made in an analogous manner to 24′:

To a solution of 24′ (50.0 mg, 0.0477 mmol) in DMF (2 mL) anddiisopropylethylamine (0.10 mL) was added Boc₂O (52.0 mg, 0.238 mmol).The reaction was stirred at room temperature for 2 h, then the reactionmixture loaded onto a 30 g C18 Isco column, eluting with 30% to 95%acetonitrile in water with 0.1% AcOH to give 29′ (42.0 mg, 0.0336 mmol,70% yield).

Compounds 30′-34′ were prepared in an analogous manner:

35′ was synthesized by charging 1.49 g Sieber amide resin (0.67 mmol/g,1.00 mmol) with FMoc-S-trityl-cysteine, and appending Fmoc-alanine,Fmoc-alanine, and Fmoc-threonine(tBu) through standard Fmoc chemistry.Cleavage of the resin with 95:3:2dichloromethane:triisopropylsilane:TFA, followed by purification bypreparative HPLC gave 35′ (34.0 mg, 0.0514 mmol, 5.1% yield).

A flask was charged with Fmoc-S-trityl-cysteine cyclohexyl amide (514mg, 0.771 mmol), and water (0.50 mL), TFA (10 mL) and triisopropylsilane(0.50 mL) were added. The reaction was stirred at room temperature for10 min, until the yellow color had faded, and all solvents were removedin vacuo. The remaining residue was dissolved in DMF (2 mL) and asolution of 2,2′-dithiodipyridine (1.03 g, 4.68 mmol) in DMF (8 mL) wasadded. 100 mM pH 7.4 phosphate buffer (2.0 mL) was added dropwise, andthe reaction was stirred at room temperature for 5 min. The reaction wasthen loaded onto a 50 g C18 Isco column, and eluting with 35% to 95%acetonitrile in water with 0.1% AcOH provided 36′ (174 mg, 0.326 mmol,42% yield).

A vial was charged with 36′ (81.0 mg, 0.152 mmol), and this wasdissolved in 4:1 DMF:piperidine (2 mL). The reaction was stirred at roomtemperature for 30 min, and all solvents were removed in vacuo. Theremaining residue was redissolved in 4:1 DMF:piperidine (2 mL), stirredat room temperature for 30 min, and again all solvents removed in vacuo.The remaining residue was dissolved in 1:1 methanol:toluene (5 mL), andall solvents removed in vacuo, to ensure complete removal of anyremaining piperidine. The crude amine 37′ was then used directly in thenext subsequent reaction.

A flask was charged with 29′ (18.4 mg, 0.0147 mmol), S-Trt cystamine(25.0 mg, 0.0783 mmol) and COMU (30.0 mg, 0.0700 mmol). DMF (5 mL) anddiisopropylethylamine (0.10 mL) were added, and the reaction stirred atroom temperature for 24 h. The reaction mixture was purified bypreparative HPLC, eluting with 5% to 95% acetonitrile in water with 0.2%AcOH, to give 38′ (10.6 mg, 0.00684 mmol, 47% yield).

Compounds 39′-50′ were made in an analogous manner to 38′.

cyclo(CYwK(Boc)TC)T-OH (51′) was synthesized by loadingFmoc-threonine(tBu) onto 2-chlorotrityl resin, and appendingFmoc-S-trityl-cysteine, Fmoc-threonine(tBu), Nα-Fmoc-Nε-Boc-lysine,Nα-Fmoc-N^(in)-Boc-D-tryptophan, Fmoc-tyrosine(tBu), andFmoc-S-trityl-cysteine using standard Fmoc chemistry. Without cleavingthe N-terminal Fmoc group, the peptide was cleaved from the resin with95:2.5:2.5 TFA:water:triisopropylsilane, cyclized with iodine in 1:1acetonitrile:water, treated with Boc₂O and diisopropylethylamine in DMF,and finally treated with 20% diethylamine in dichloromethane, andpurified by reverse phase chromatography to provide 51′.

Compounds 52′ and 53′ were made in an analogous manner to 51′.

To a solution of octreotide acetate (545 mg, 0.505 mmol) in DMF (10 mL)was added diisopropylethylamine (0.50 mL). The solution was then cooledto −40° C., and a solution of BocOSu (119 mg, 0.553 mmol) in DMF (5 mL)was added dropwise. The reaction was stirred at −40° C. for 1 h, thengradually warmed up to room temperature over 1 h. Most of the DMF wasremoved in vacuo, and the remaining residue was loaded onto a 50 g C18Isco column. Elution with 15% to 60% acetonitrile in water with 0.1%AcOH gave Lys-Boc octreotide acetate (54′, 440 mg, 0.373 mmol, 74%yield) with >95% regioselectivity by ¹H NMR.

Compound 55′, Lys-Boc vapreotide, was made in an analogous manner to54′.

Fmoc-aspartic acid(tBu) was loaded onto 2-chlorotrityl resin, andFmoc-aspartic acid(tBu), Fmoc-S-trityl-cysteine, and Ac₂O were appendedusing standard Fmoc chemistry. The peptide was cleaved from the resin,and purified by reverse phase chromatography. The purified peptide (50mg, 0.067 mmol) was dissolved in dichloromethane (3 mL), and DCC (20.6mg, 0.100 mmol) and HOSu (11.5 mg, 0.100 mmol) were added, and thereaction stirred overnight. The resulting solution was filtered, thefiltrate concentrated in vacuo, and the crude NHS ester used as is.

A flask was charged with triphenylmethanethiol (1.23 g, 4.45 mmol) anddichloromethane (7 mL), diisopropylethylamine (1.0 mL) and acrolein(0.60 mL, 8.98 mmol) were added. The reaction was stirred at roomtemperature for 1 h, and all solvents were removed in vacuo to givetrityl 3-mercaptopropionaldehyde (57′, 1.48 g, 4.45 mmol) which was usedcrude in the next step.

Compounds 58′ and 59′ were made in an analogous manner to compound 57′.

A flask was charged with ethyl 1-piperazinylacetate (1.03 g, 6.02 mmol)and trityl 3-mercaptopropionaldehyde (2.00 g, 6.02 mmol). The reagentswere dissolved in dichloromethane (20 mL), acetic acid (0.10 mL) wasadded, and sodium triacetoxyborohydride (3.19 g, 15.0 mmol) was added.The reaction was stirred at room temperature for 2 h, then purified bysilica gel chromatography to provide 60′ (1.80 g, 3.69 mmol, 61% yield).

60′ (640 mg, 1.31 mmol) was dissolved in 10:1 ethanol:water (10 mL), andlithium hydroxide (63.0 mg, 2.62 mmol) was added. The reaction wasstirred at room temperature for 2 h, then acidified with 10% citricacid. The resulting solid was filtered, washed with water, and dried toprovide 61′ (400 mg, 0.870 mmol, 66% yield).

61′ (76.3 mg, 0.167 mmol) was dissolved in dichloromethane (2 mL). DCC(51.5 mg, 0.250 mmol) and HOSu (28.8 mg, 0.250 mmol) were added, thereaction stirred overnight, then the reaction was filtered and thefiltrate concentrated in vacuo. The resulting NHS ester was used crudein the next step.

To a solution of 61′ (450 mg, 0.978 mmol) in dichloromethane (15 mL) wasadded CDI (190 mg, 1.17 mmol) as a solid. The reaction was stirred atroom temperature for 30 min, then N,O-dimethylhydroxylaminehydrochloride (114 mg, 1.17 mmol) was added as a solid. The reaction wasstirred at room temperature for 4 h, the reaction washed with water (15mL), and the organic layer dried and concentrated in vacuo to give 63′(200 mg, 0.398 mmol, 41% yield).

To a solution of 63′ (50 mg, 0.099 mmol) in THF (5 mL), at 0° C., wasadded a solution of lithium aluminum hydride (0.13 mL, 1M in THF, 0.13mmol). The reaction was stirred at 0° C. for 2 h, then quenched by 1 NHCl (5 mL) and extracted with ethyl acetate (10 mL). The organic layerwas dried with MgSO₄, and the concentrated in vacuo to provide crude 64′(20 mg, 0.045 mmol, 45% yield), which was used crude in the next step.

Compound 65′ was made in an analogous manner to compound 64′.

A flask was charged with 66′ (200 mg, 0.388 mmol), and this wasdissolved in 4:1 DMF:diethylamine (10 mL). The reaction was stirred atroom temperature for 4 h, and the solvent removed in vacuo. The residuewas dissolved in a few drops of dichloromethane, and diethyl ether (20mL) was added to precipitate the product. The crude 67′ was filtered,and taken directly on to the next step.

The crude 67′ was dissolved in DMF (5 mL), and trityl3-mercaptopropionic acid NHS ester (173 mg, 0.388 mmol) was added,followed by diisopropylethylamine (0.30 mL). The reaction was stirred atroom temperature for 3 h, and the reaction was purified by preparativeHPLC to provide 68′ (140 mg, 0.224 mmol, 58% yield).

A vial was charged with 68′ (40 mg, 0.064 mmol), DCC (14 mg, 0.064 mmol)and HOSu (7.4 mg, 0.064 mmol). Dichloromethane (2 mL) was added, thereaction stirred at room temperature for 16 h, and the reactionfiltered, the filtrate collected and concentrated in vacuo, and thecrude 69′ used directly in the next step.

A flask was charged with N-Boc D-tyrosine methyl ester (1.00 g, 3.39mmol), triphenylphosphine (977 mg, 3.73 mmol), andS-trityl-2-mercaptoethanol (1.08 g, 3.39 mmol). While under nitrogen,THF (20 mL) was added, followed by dropwise addition ofdiethylazodicarboxylate (0.64 mL, 4.1 mmol). The reaction was stirred atroom temperature for 16 h, and the solvent removed in vacuo, and theremaining residue purified by preparative HPLC to provide 70′ (880 mg,1.48 mmol, 44% yield).

70′ (880 mg, 1.48 mmol) was dissolved in ethanol (20 mL) and water (2mL), and lithium hydroxide (72 mg, 3.0 mmol) was added. The reaction wasstirred at room temperature for 3 h, and the reaction purified bypreparative HPLC to provide 71′ (820 mg, 1.41 mmol, 95% yield).

A vial was charged with 71′ (58 mg, 0.10 mmol), DCC (21 mg, 0.10 mmol)and HOSu (11 mg, 0.10 mmol). Dichloromethane (2 mL) was added, thereaction stirred at room temperature for 16 h, then filtered. Thefiltrate was concentrated in vacuo, and the collected crude 72′ was useddirectly in the next step.

To a solution of 52′ (62.0 mg, 0.0539 mmol) in DMF (3 mL) was addedtrityl 3-mercaptopropionic acid NHS ester (90.0 mg, 0.202 mmol).Diisopropylethylamine (0.20 mL) was added, and the reaction stirred atroom temperature for 24 h. The reaction mixture was then loaded onto a30 g C18 Isco column, and eluting with 5% to 95% acetonitrile in waterwith 0.1% AcOH provided 73′ (43.1 mg, 0.0291 mmol, 54% yield).

Compounds 74′-85′ were made in an analogous manner to 73′.

54′ (413 mg, 0.350 mmol) was dissolved in dichloromethane (10 mL),methanol (3 mL) and acetic acid (0.25 mL). 57′ (180 mg, 0.541 mmol) wasadded, followed by sodium triacetoxyborohydride (115 mg, 0.541 mmol).The reaction was stirred at room temperature for 2 h, and all solventswere removed in vacuo. The remaining residue was dissolved in a minimalamount of DMF, and loaded onto a 50 g C18 Isco column. Eluting with 15%to 85% acetonitrile in water with 0.1% TFA provided 86′ as thetrifluoroacetate salt (389 mg, 0.251 mmol, 72% yield).

Compounds 87′-94′ were made in an analogous manner to 86′:

To a solution of p-nitrophenyl chloroformate (145 mg, 0.719 mmol) indichloromethane (1 mL) and diisopropylethylamine (0.20 mL) was added asolution of S-trityl cystamine (168 mg, 0.526 mmol) in dichloromethane(2 mL). The reaction was stirred at room temperature for 5 min, and thereaction mixture loaded directly onto a 24 g silica gel column. Elutingwith 0% to 30% ethyl acetate in heptane provided 95′ (120 mg, 0.247mmol, 47% yield).

To a solution of 54′ (162 mg, 0.137 mmol) in THF (3 mL) anddiisopropylethylamine (0.50 mL) was added a solution of 95′ (120 mg,0.247 mmol) in THF (1 mL). DMAP (36.6 mg, 0.300 mmol) was added as asolid, and the reaction stirred at 50° C. for 6 h. All solvent wasremoved in vacuo, and the remaining material loaded onto a 30 g C18 Iscocolumn, eluting with 15% to 95% acetonitrile in water with 0.1% AcOH toprovide 96′ (201 mg, 0.137 mmol, 100% yield).

A vial was charged with 88′ (30.0 mg, 0.0190 mmol), COMU (16.0 mg,0.0.0374 mmol), and 11′ (35.0 mg, 0.0896 mmol). Dichloromethane (2 mL)and diisopropylethylamine (0.20 mL) were added, and the reaction stirredat room temperature for 20 h. The solvent was removed in vacuo, and theremaining material loaded onto a 30 g C18 Isco column, and eluting with40% to 95% acetonitrile in water with 0.1% AcOH provided 97′ (16.6 mg,0.00903 mmol, 47% yield).

S-trityl-L-cysteine ethylenediamine amide (40.0 mg, 0.0986 mmol) andglutaric anhydride (45.0 mg, 0.395 mmol) were dissolved in DMF (2 mL).The reaction was stirred at room temperature for 16 h, and the reactionmixture purified by preparative HPLC to provide 98′ (30.0 mg, 0.0473mmol, 48% yield).

A vial was charged with 98′ (8.0 mg, 0.0126 mmol), 54′ (28.2 mg, 0.0252mmol), EDC (5.8 mg, 0.030 mmol) and HOBt (4.1 mg, 0.30 mmol). DMF (2 mL)and diisopropylethylamine (9 μL, 0.05 mmol) were added, and the reactionstirred at 35° C. for 16 h. The reaction was then purified bypreparative HPLC to give 99′ (21.0 mg, 0.00740 mmol, 59% yield).

Synthesis of DM1 Conjugates

A flask was charged with DM-1 (41.8 mg, 0.0566 mmol), anddichloromethane (2 mL), diisopropylethylamine (0.10 mL) and acrolein(0.25 mL) were added. The reaction was stirred at room temperature for 5min, and all solvents were removed in vacuo. The remaining residue wasredissolved in dichloromethane (1 mL) and toluene (0.5 mL), and thesolvents removed in vacuo again, to ensure complete removal of anyremaining acrolein. To the remaining residue was added a solution of 54′(61.0 mg, 0.0517 mmol) in dichloromethane (2 mL) and acetic acid (0.05mL). Sodium triacetoxyborohydride (11.5 mg, 0.0523 mmol) was added as asolid, and the reaction stirred at room temperature for 1 h. All solventwas removed in vacuo, and the remaining residue was dissolved in TFA (3mL). The reaction was stirred at room temperature for 5 min, and most ofthe TFA was removed in vacuo. The remaining material was purified bypreparative HPLC (5% to 55% acetonitrile in water with 0.2% AcOH) toprovide Conjugate 100 as the acetate salt (7.6 mg, 0.0040 mmol, 7.6%yield). LCMS M/Z: 899.0 [(M+2)/2].

To a solution of 2,2′-dithiodipyridine (1.24 g, 5.65 mmol) in DMF (8 mL)and diisopropylethylamine (1 mL) was added a solution of DM-1 (417 mg,0.565 mmol) in 2 mL DMF, dropwise over 5 min. The reaction was stirredat room temperature for another 30 min, and the reaction mixture loadedonto a C18 Isco gold column. Eluting with 25% to 85% acetonitrile inwater provided DM1-SSPy (287 mg, 0.339 mmol, 60% yield). LCMS M/Z: 847.3[M+1].

A vial was charged with 4′ (20.0 mg, 0.0209 mmol), and a solution ofDM-1/SSPy (17.7 mg, 0.0209 mmol) in DMF (2 mL) was added. 100 mM pH 7.4phosphate buffer (1.0 mL) was added dropwise while stirring rapidly, andthe reaction stirred for another 5 min at room temperature. The reactionmixture was purified by preparative HPLC (5% to 65% acetonitrile inwater with 0.2% AcOH) to provide Conjugate 10 (22.1 mg, 0.0126 mmol, 60%yield) as the acetate salt. LCMS M/Z: 837.5 [(M+2-H₂O)/2].

DM-1 conjugation method A: A vial was charged with 8′ (20.2 mg, 0.0159mmol), and water (0.025 mL), TFA (1 mL) and triisopropylsilane (0.025mL) were added. The reaction was stirred at room temperature for 5 min,and all solvents were removed in vacuo. To the remaining residue wasadded a solution of DM-1/SSPy (13.5 mg, 0.0159 mmol) in DMF (3 mL) wasadded. While stirring, 100 mM pH 7.4 phosphate buffer (1 mL) was addeddropwise, and the reaction stirred for an additional 5 min at roomtemperature. The reaction was then acidified with acetic acid (0.25 mL).The reaction mixture was then purified by preparative HPLC (5% to 70%acetonitrile in water with 0.2% AcOH) to provide Conjugate 14 (9.3 mg,0.0054 mmol, 34% yield) as the acetate salt. LCMS M/Z: 832.3 [(M+2)/2].

DM-1 conjugation method B: A vial was charged with 22′ (21.2 mg, 0.0177mmol) and TFA (1 mL) was added. The reaction stirred at room temperaturefor 5 min, and all solvent was removed in vacuo. To the remainingresidue was added a solution of DM-1 (13.1 mg, 0.0177 mmol) in DMF (2mL). While stirring, 100 mM pH 7.4 phosphate buffer (1 mL) was addeddropwise, and the reaction then stirred at room temperature for another5 min. The reaction was acidified by adding acetic acid (0.25 mL), andthe reaction mixture purified by preparative HPLC (5% to 75%acetonitrile in water with 0.2% AcOH) to yield Conjugate 22 (20.2 mg,0.0114 mmol, 64% yield) as the acetate salt. LCMS M/Z: 860.5 [(M+2)/2].

DM-1 conjugation method C: A vial was charged with 7 (25.7 mg, 0.0233mmol), and TFA (1 mL) was added. The reaction was stirred at roomtemperature for 5 min, and all solvent was removed in vacuo. To theremaining residue was added a solution of DM-1 (17.2 mg, 0.0233 mmol) inDMF (4 mL), followed by diisopropylethylamine (0.25 mL). The reactionwas stirred at room temperature for 10 min, and was then acidified byadding acetic acid (0.40 mL). The reaction mixture was then purified bypreparative HPLC (5% to 70% acetonitrile in water with 0.2% AcOH) toprovide Conjugate 12 (25.6 mg, 0.0142 mmol, 61% yield) as the acetatesalt. LCMS M/Z: 872.0 [(M+2)/2].

Synthesis of 57A. Fmoc-Cysteine(Trt)-OH was loaded onto 2-chlorotritylresin (25.0 g resin, 100-200, 1 meq/g loading). Iterative deprotectionwith 4:1. DMF:piperidine, and coupling subsequently withFmoc-Threonine(tBu)-OH, Nα-Fmoc-Nε-Boc-lysine,Na-Fmoc-N^(in)-Boc-D-tryptophan, Fmoc-tyrosine(tBu),Fmoc-Cysteine(Trt)-OH, and Boc-D-phenylalanine using standard SPPSconditions (Nature Prot. 2012, 432) gave the linear peptide bound to theresin. 8 g of resin (0.338 mmol/g) was stirred with DMF (80 mL)containing iodine (3 equiv.) for 3 h at rt, then filtered and washedwith DMF (2×40 mL). The resin was resubmitted with iodine (3 equiv.) inDMF (80 mL) for 3 h. Resin was washed with DMF (2×40 mL), then DCM (2×40mL) and resin was dried in vacuo at rt.

Resin (8 g, 0.338 mmol/g) was swelled in DCM (80 mL) andhexafluoroisopropanol (30 mL) was added at rt over ˜1 min. The resultingmixture was stirred for 30 min at rt, then filtered and washed with DCM(2×40 mL). The filtrate was concentrated in vacuo at 20-25 C. The resinwas resubmitted to cleavage conditions with DCM (80 mL) andhexafluoroisopropanol (30 mL), stirred for 30 min, then filtered andwashed with DCM (2×40 mL). The filtrate was evaporated under reducedpressure at 20-25° C. Crude residue was dissolved in MTBE (minimumamount), then added dropwise to n-heptane stirred at rt to produce aprecipitate. The solid was filtered and dried at rt to yield 57A (2.95g, 2.17 mmol, 80% yield). LCMS M/Z: 1361 [M+1].

100 mL RBF was charged with 57A (2.73 g, 2.01 mmol) and(2R)-2-amino-3-tritylsulfanyl-propanamide (728.60 mg, 2.01 mmol).Dichloromethane (27.00 mL) was added followed by diisopropylethylamine(519.54 mg, 4.02 mmol) and HATU (840.69 mg, 2.21 mmol). After 1.5 h,conversion was complete by HPLC/MS. 27 g silica gel (10 weighs) wascharged in a fritted glass funnel. 40:60 TBME/DCM was used to wetsilica. The DCM solution was added on top of the silica gel, then elutedwith 40:60 TBME/DCM (250 mL), followed by 2% isopropanol in 40:60TBME/DCM (250 mL). The filtrate was evaporated. The desired product 57Bwas obtained. (3.16 g, 92% yield). LCMS M/Z: 1705 [M+1].

Deprotection method A. 57B (500.00 mg, 293.23 umol) was charged in a 50mL RBF, 2,2′-dithiodipyridine (129.85 mg, 589.39 umol) andtriisopropylsilane (386.81 mg, 2.44 mmol) were added, followed byhexafluoroisopropanol (8 mL), then by 8 mL of 0.5 M HCl inhexafluoroisopropanol (8 mL). After 2 h, HPLC/MS showed completeconversion. Solution was added slowly to TBME (30 mL) and theprecipitate formed was filtered, then washed with TBME (30 mL). Thesolid was dissolved in 1 M AcOH (8 mL) and the solution was stirred atRT for 2 h. HPLC/MS showed complete conversion to desired product. Thecrude mixture was directly injected on a 100 g C18 Isco gold column.Column was flushed with 360 mL (3 volumes) 100 mM ammonium acetate, thenre-equilibrated with 5% acetonitrile in water with 0.1% AcOH (2 volumes)and eluted using gradient of 5% to 30% acetonitrile in water with 0.1%AcOH for 24 mins. Pure fractions were lyophilized to give 57C as theacetate salt. LCMS M/Z: 1160 [M+1].

Deprotection method B. Flask was charged with 57C (502 mg, 0.294 mmol).In a separate vial was charged TFA (8.8 mL), triisopropylsilane (0.20mL), water (0.50 mL), and thioanisole (0.50 mL). This vial was shakenuntil the mixture turned homogeneous, then the deprotection cocktail wasadded to the solid 57C. The flask was stirred until everything went intosolution, then stirred at room temperature for another 30 min. HPLC/MSshows complete deprotection. TFA was removed in vacuo, until volume ofreaction mixture ca. 1 mL. Ethanol (30 mL) was added to the reaction,solution was cooled to 0° C., then a solution of 2,2′-dithiodipyridine(130 mg) in 10 mL ethanol was added. Solution was stirred at 0° C. for 1h, then stirred at room temperature for 2 h. HPLC/MS showed completeconversion to SSPy product. Pyridine (5 mL) was added, solution wasstirred at room temperature for 5 min, then all solvents were removed invacuo. Residue was dissolved in 2 mL DMF and 8 mL 1% AcOH in water,loaded onto a 100 g C18 Isco gold column. Column was flushed with 360 mL(3 volumes) 100 mM ammonium acetate, then re-equilibrated with 5%acetonitrile in water with 0.1% AcOH (2 volumes) and eluted usinggradient of 5% to 30% acetonitrile in water with 0.1% AcOH for 24 mins.Pure fractions were lyophilized to give 57C as the acetate salt. 241 mgisolated (61.5% yield). LCMS M/Z: 1160 [M+1].

A 100 mL RBF was charged with 57C bis-acetate salt (210 mg, 0.164 mmol)and this was dissolved in THF (4 mL) and 0.2M AcOH (3.6 mL) and 0.2MNaOAc (0.4 mL). 57C was checked by LCMS by taking an aliquot of 2 uL anddissolving in 50 uL methanol, and running on the method below. To thereaction mixture was added a solution of DM-1 (124 mg, 0.167 mmol) inTHF (4 mL). The reaction was stirred at room temperature for 1 h. A 2 uLaliquot was removed, diluted with 50 uL methanol, and checked by LCMS.Area of BT-891:area of BT-976 is >10:1 at 280 nm, and the reaction isjudged complete. All solvents were removed in vacuo, with bathtemperature at 35° C., and at 10 mbar for 45 min to remove all water.The remaining residue was dissolved in 1 mL DMF, and this was dilutedwith 3 mL aqueous 1% acetic acid. This solution was loaded onto a 50 gRediSep Rf Gold C18 column (20-40 micron particle size). Another 1 mLDMF and 3 mL 1% acetic acid was added to the reaction flask, and thiswas also loaded onto the C18 column. Column eluted with 40 mL/mingradient, 17 min run. 2 min @ 5% acetonitrile in water with 0.1% AcOH,then 15 min gradient from 5% to 40% acetonitrile in water with 0.1%AcOH. Product elutes as a single peak around 35% acetonitrile. Elutedfractions collected, most of the solvent removed in vacuo until totalvolume was 5 mL. This solution was transferred to an amber vial, and 5mL of a 1:1 mix of acetonitrile:water was used to rinse the flask, andtransferred to the amber vial. 2 uL aliquot was diluted with 50 uLmethanol, checked by LCMS method below. The resulting solution wasfrozen by placing in a dry ice/acetone bath, and dried in a benchtopfreeze drier at 200 millitorr for 3 d. Isolated 57 as the bis-acetatesalt (262 mg, 0.137 mmol, 84% yield). LCMS M/Z: 893.4 [(M+2)/2].

TABLE 13 Synthesis of DM1 conjugates DM-1 conjugation Final Compoundmethod conjugate LCMS M/Z  9′ A 16  910.2 [(M + 2)/2] 14′ A 18  948.0[(M + 2)/2] 15′ A 24  868.0 [(M + 2)/2] 16′ A 26  880.9 [(M + 2)/2] 17′A 30  860.8 [(M + 2)/2] 20′ C 20  916.0 [(M + 3)/3] 21′ C 32 1030.4[(M + 2)/2] 23′ B 28  852.5 [(M + 2)/2] 38′ A 35  924.3 [(M + 2)/2] 39′A 37  942.3 [(M + 2)/2] 40′ A 39 1015.0 [(M + 2)/2] 41′ C 41  962.0[(M + 2)/2] 42′ A 43  958.0 [(M + 2)/2] 43′ A 45 1001.0 [(M + 2)/2] 44′A 47 1024.0 [(M + 2)/2] 45′ A 49  965.0 [(M + 2)/2] 46′ A 51  977.5[(M + 2)/2] 47′ A 57  893.4 [(M + 2)/2] 48′ B 59  871.9 [(M + 2)/2] 49′B 63  934.5 [(M + 2)/2] 50′ B 65  957.8 [(M + 2)/ 2] 73′ A 74  937.0[(M + 2)/2] 74′ A 68 n.d. 75′ A 70  978.0 [(M + 2)/ 2] 76′ A 72 1065.5[(M + 2)/2] 77′ A 86  977.9 [(M + 2)/2] 78′ A 98 1001.5 [(M + 2 −H₂O)/2] 79′ C 104  979.5 [(M + 2 − H₂O)/2] 80′ C 108  938.5 [(M + 2 −H₂O)/2] 81′ A 119  871.1 [(M + 2)/2] 82′ A 111  930.9 [(M + 2)/2] 83′ A113  927.4 [(M + 2)/2] 84′ A 115  951.5 [(M + 2)/2] 85′ C 117 n.d. 86′ A76  915.0 [(M + 2)/2] 87′ A 78  971.9 [(M + 2)/2] 88′ A 80  930.0 [(M +2)/2] 89′ A 82  921.9 [(M + 2)/2] 90′ C 90  940.5 [(M + 2)/2] 91′ A 94 971.0 [(M + 2)/2] 92′ A 96 1003.0 [(M + 2)/2] 93′ A 102 1041.6 [(M +2)/2] 94′ A 106  985.0 [(M + 2)/2] 96′ A 84  965.0 [(M + 2)/2] 97′ A 88n.d. 99′ A 92 1044.1 [(M + 3)/3]

Example 15: Nanoparticle Formulation of Drug Conjugate

Nanoparticle formulation of a typical conjugate X, which may be anyconjugate of the present invention. Conjugate X was successfullyencapsulated in polymeric nanoparticles using a single oil in wateremulsion method (refer to Table 14 below). In a typical water-emulsionmethod, the drug conjugate and a suitable polymer or block copolymer ora mixture of polymers/block copolymers, were dissolved in organicsolvents such as dichloromethane (DCM), ethyl acetate (EtAc) orchloroform to form the oil phase. Co-solvents such as dimethyl formamide(DMF) or acetonitrile (ACN) or dimethyl sulfoxide (DMSO) or benzylalcohol (BA) were sometimes used to control the size of thenanoparticles and/or to solubilize the drug conjugates. A range ofpolymers including PLA97-b-PEG5, PLA35-b-PEG5 and PLA16-b-PEG5copolymers were used in the formulations. Surfactants such as Tween® 80,sodium cholate, Solutol® HS or phospholipids were used in the aqueousphase to assist in the formation of a fine emulsion. The oil phase wasslowly added to the continuously stirred aqueous phase containing anemulsifier (such as Tween 80) at a typical 10%/90% v/v oil/water ratioand a coarse emulsion was prepared using a rotor-stator homogenizer oran ultrasound bath. The coarse emulsion was then processed through ahigh-pressure homogenizer (operated at 10,000 psi) for N=4 passes toform a nanoemulsion. The nanoemulsion was subsequently quenched by a10-fold dilution with cold (0-5° C.) water for injection quality waterto remove the major portion of the ethyl acetate solvent in thenanoemulsion droplet, resulting in hardening of the emulsion dropletsand formation of a nanoparticle suspension. In some cases, volatileorganic solvents such as dichloromethane can be removed by rotaryevaporation. Tangential flow filtration (500 kDa MWCO, mPES membrane)was used to concentrate and wash the nanoparticle suspension with waterfor injection quality water (with or without surfactants/salts). Thefree drug conjugate was removed from the nanosuspension using a varietyof techniques. A cryoprotectant serving also as tonicity agent (e.g.,10% sucrose) was added to the nanoparticle suspension and theformulation was sterile filtered through a 0.22 μm filter. Theformulation was stored frozen at ≤−20° C. Particle size (Z-ave) and thepolydispersity index (PDI) determined by dynamic light scattering of thenanoparticles were characterized by dynamic light scattering, assummarized in the table below. The actual drug load was determined usingHPLC and UV-visible absorbance. This was accomplished by evaporating thewater from a known volume of the nanoparticle solution and dissolvingthe solids in an appropriate solvent such as DMF. The drug concentrationwas normalized to the total solids recovered after evaporation.Encapsulation efficiency was calculated as the ratio between the actualand theoretical drug load.

Formulations Using Hydrophobic Ion-Pairing (HIP) of Conjugate X

In some instances, HIP techniques were used to enhance the lipophilicityof conjugate X. The conjugate X has one or more positively chargedmoieties. A negatively charged counter-ion such as dioctyl sodiumsulfosuccinate (AOT) molecules was used for everyone molecule of theconjugate to form the HIP. The conjugate X and the AOT were added to amethanol, dichloromethane and water mixture and allowed to shake for 1hour. After further addition of dichloromethane and water to thismixture, the X/AOT HIP was extracted from the dichloromethane phase anddried. In some embodiments, DMF was used to solubilize the HIP complex.The results of the formulations are summarized in Table 14 below.

TABLE 14 Formulations of conjugates 76, 10 and 78 Drug Conjugate 76 1078 NP3-Efficacy NP6-Efficacy NP05-Efficacy Process Single emulsionSingle emulsion Single emulsion Polymer PLA35-mPEG5 PLA35-mPEG5/PLA50-PLA35- Me (50%/50%) mPEG5/PLA50-Me (50%/50%) Polymer 100 100 100concentration, mg/mL Emulsion Volume, 100 200 200 mL Oil phase 87.5%EA/12.5% DMF/ 43.05% EA/ 42.5% EA/ 13.9% DMF/43.05% DCM 15% DMF/42.5%DCM Drug 76:AOT 10 78 Aqueous phase 0.1% Tween 80/Water 0.05%DiOctPC/Water  0.05% DiOctPC/Water Oil phase volume 10.00% 10.00% 10.00%fraction, % Wash Wash 12X with Saline; Wash 4X with cold water; Wash 6Xwith cold 5X cold water; Quench Quench 7X in 30oC water; Quench 10X 7Xin 30oC water, wash water, wash 21X in ice in 30oC water, wash 5X in icecold water; cold water; drug 25X in ice cold extraction water; drugextraction Z.ave/PDI 72.7/0.057 129.00 137.50 (quenched Emulsion)Z.ave/PDI (post TFF 67.16/0.04 115.7/0.095 119.1/0.044 filtered) TDL (wt%) 6.09 3.91 4.93 ADL (wt %) 4.70 0.48 1.04 EE = ADL/TDL, % 0.772 0.1220.211 Potency, mg/mL 1.214 0.2763 0.363 TDL: Theoretical Drug LoadingADL: Actual Drug Loading NA: not available EE: encapsulation efficiency

These data demonstrate that conditions can be invented for the efficientencapsulation of conjugate X in nanoparticles.

Example 16: IC₅₀ in H524 Cells

Conjugates of the present invention were assessed in an in vitro assayevaluating inhibition of cell proliferation. NCI-H524 (ATCC) human lungcancer cells were plated in 96 well, V-bottomed plates (Costar) at aconcentration of 5,000 cells/well. 24 hours later, cells were treatedwith either conjugate for. 2 hours and further incubated 70 hours, orfor octreotide competition experiments, treated with 100 μM octreotidefor 30 min, and then treated with conjugate for 2 hours, and furtherincubated for 70 hours. Conjugate starting dose was 20 μM and three foldserial dilutions were done for a total of ten points. After 2 hours oftreatment, cells were spun down, the drug containing media was removed,and fresh complete medium was added and used to resuspend the cells,which were spun again. After removal of the wash media, the cells wereresuspended in complete medium, then transferred into white walled, flatbottomed 96 well plates. Cells were further incubated for an additional70 hours to measure inhibition of cell proliferation. Proliferation wasmeasured using CellTiter Glo reagent using the standard protocol(Promega) and a Glomax multi+detection system (Promega). Percentproliferation inhibition was calculated using the following formula: %inhibition=(control-treatment)/control*100. Control is defined asvehicle alone. IC50 curves were generated using the nonlinear regressionanalysis (four parameter) with GraphPad Prism 6. IC₅₀ values for theconjugates comprising DM1 were shown in Table 15 below. These datademonstrate that conjugates retain the ability to bind to somatostatinand internalize the receptor. In some instances, this also shows thatthe linker is cleaved to activate the cytotoxic payload effectively tokill the tumor cells.

Example 17: Activity Dependence on the Receptor

Conjugates of the present invention were tested for their activitydependence on the somatostatin receptor. Active agent Z in the conjugatewas selected from auristatin, carbazitaxel, DM1, doxorubicin, platinum,SN-38 and vinblastine. Active agent Z is connected to octreotide withvarious linkers. Proliferation IC₅₀, values of the conjugates weremeasured. Proliferation IC₅₀ values of the conjugates without octreotidecompetition were also measured. The ratios of IC₅₀ with octreotidecompetition and IC₅₀ without octreotide competition were shown in FIG.3. The ratio of IC₅₀ with octreotide competition and IC₅₀ withoutoctreotide competition is an indicator of whether activity is at leastpartially dependent on bind to the somatostatin receptor. Conjugatescomprising DM1 showed a ration of more than 1, indicating lower IC₅₀,i.e., better efficacy, without octreotide competition than withoctreotide competition. Therefore, the activity of conjugates comprisingDM1 is dependent on the binding to the somatostatin receptor.

IC₅₀ values for the conjugates comprising DM1 with octreotidecompetition were shown in Table 15 below. The results in Table 15 showedIC₅₀ values for all the conjugates comprising DM1 increased withoctreotide competition, which means the efficacy of the conjugatescomprising DM1 decreased with octreotide competition. DM1 alone did notshow such a change in IC₅₀ values with octreotide competition.Therefore, the efficacy of conjugates comprising DM1 at least partiallydepends on the binding of the conjugates to the somatostatin receptor.

Example 18: H69 Tumor DM-1 Levels

To examine the ability of conjugates of the present invention toaccumulate in tumors, a murine cancer model was used. All mice weretreated in accordance with the OLAW Public Health Service Policy onHuman Care and Use of Laboratory Animals and the ILAR Guide for the Careand Use of Laboratory Animals. All in vivo studies were conductedfollowing the protocols approved by the Blend Therapeutics InstitutionalAnimal Care and Use Committee. Animals were inoculated with2.5×10{circumflex over ( )}6 NCI-H69 SCLC (small cell lung cancer) cellsin 1:1 RPMI 1640 (Invitrogen, Carlsbad, Calif.)/Matrigel® (BDBiosciences, San Jose, Calif.) via subcutaneous injection to the rightflank. Tumors were allowed to reach an approximate volume of ˜500 mm3.Animals were then randomized into treatment groups of 3 animals per timepoint and were dosed at 1 mg/kg (10% propylene glycol in water forinjection for free conjugates, or 10% sucrose for nanoparticles), or 0.4mg/kg for DM-1 (10% propylene glycol in water for injection). The 24hour time point was used as a benchmark across conjugates.

Tumor DM-1 levels were determined by liquid chromatography massspectrometry (LC/MS-MS). Four volumes of 5 mM 6-maleimidohexanoic acidto 1 part tumor v/w was added and homogenized for about 10-15 secondswith a handheld homogenizer. 10 μL of 500 mMTris(2-carboxyethyl)phosphine was added to 100 uL of tumor homogenate,mixed well and incubated at room temperature for about 5-15 min. 200-300uL of acetonitrile was used to precipitate proteins in tumor homogenate,samples were centrifuged for 5 min, and supernatant was injected ontoLC/MS-MS system for DM-1 analysis. H69 tumor DM-1 levels were shown inTable 15.

TABLE 15 H524 cell assay and in vivo tumor uptake results for DM1conjugates H524 IC₅₀ ₊ 100 uM H69 tumor DM-1 Conjugate H524 IC₅₀ (nM)octreotide (nM) levels (nM)  4 367 853 n.d.  6 234 629 n.d.  7 955 1770n.d.  68 821 1340 n.d.  70 317 417 n.d.  72 n.d. n.d. n.d.  74 549 562n.d.  76 120 768 62.2  76 NP7 n.d. n.d. 221  10 156 625 72.1  10 NP6n.d. n.d. 883  35 253 1000 23.2  78 124 365 36.2  78 NP5 n.d. n.d. 223 80 152 157 91.2  82 148 867 n.d.  84 76 312 n.d.  37 44 166 n.d. 111123 742 82.6  86 386 553 n.d. 113 1080 4920 n.d. 115 327 1495 37.1  392642 3578 11.8  88 80 180 n.d.  41 86 1343 n.d.  90 84 1021 n.d.  92 367471 n.d.  94 285 1017 n.d.  96 206 397 n.d.  98 690 1121 n.d. 100 3031055 n.d. 117 6 54 n.d. 102 335 579 n.d.  43 1165 2046 n.d.  45 345 481n.d.  47 2129 1267 n.d.  49 69 243 n.d.  51 124 238 n.d.  12 1685 2345n.d. 104 279 705 n.d.  14 298 877 70.1 106 400 666 n.d.  16 223 398 96.4 53 42 175 n.d.  18 1480 2160 86.3  20 241 427 n.d. 108 96 1280 n.d.  22332 741 145  24 149 982 n.d.  26 787 1161 n.d.  55 47 395 95.3  28 6691000 n.d.  57 136 1654 243  59 24 170 n.d.  30 470 2017 n.d.  32 310 733n.d.  61 89 274 n.d.  63 44 168 n.d.  65 30 229 n.d. 119 2656 >5000 n.d.DM-1 90 90 31.6

Example 19: Effect of Conjugate 10 and Conjugate 10 NP6 Compounds onTumor Growth and Pharmacokinetics Studies

Applicants assessed the activity of a conjugate and a nanoparticleformulation of the conjugate in vivo. In these experiments, the abilityof compounds to affect the growth of human NCI-H69 SCLC was tested. Forthe in vivo study, 8 week old female NCR nude mice were inoculatedsubcutaneously into the right flank with 2 million cells in 1:1 RPMI1640 (Invitrogen, Carlsbad, Calif.)/Matrigel® (BD Biosciences, San Jose,Calif.). Tumor measurements were taken twice weekly, using verniercalipers. Tumor volume was calculated using the formula:V=0.5×width×width×length.

When tumors approached a volume of 200 mm³, mice were randomized intofour groups of ten animals. Mice were treated with vehicle control (10%propylene glycol in water for injection), Conjugate 10 at 2 mg/kg (10%propylene glycol in water for injection), Conjugate 10 NP6 nanoparticleat 2 mg/kg (10% Sucrose), or DM1 at 0.8 mg/kg (10% propylene glycol inwater for injection). Mice were dosed once weekly for two doses. Finaltumor volumes were analyzed using with a one-way analysis of varianceand Tukey multiple comparison test. Tumor volumes were tracked over acourse of up to 100 days as shown in FIG. 4.

As shown in FIG. 4, the tumor volume increased rapidly for vehicle andDM1 controls. Conjugate 10 alone initially provided tumor regression,but tumors regrew over the course of the study. Conjugate 10 NP6provided complete cures, i.e., 9 out of 9 mice were tumor-free at 100days post dosing after only two doses on days 1 and 8. A Kaplan-Meriertumor volume curve of percentage of mice with tumor size less than 2000mm³ in FIG. 5 shows that out to 100 days no animals have come off of thestudy in the Conjugate 10 NP6 group, but in the Conjugate 10 alone groupthree animals had to be removed from the study due to large tumor size.

Tumor volume study was repeated with three doses for vehicle, Conjugate10 (0.7 mg/kg each), and Conjugate 10 NP6 (0.7 mg/kg each). Tumorvolumes were tracked for 30 days. The results were shown in FIG. 6.Conjugate 10 NP6 again showed significantly superior efficacy to freeConjugate 10.

Pharmacokinetics studies in rat plasma were also carried out. Rat plasmapK of Conjugate 10 and Conjugate 10 NP6 was shown in FIG. 7. AUC valueswere show in Table 16. Incorporating Conjugate 10 in a nanoparticleincreases AUC for Conjugate 10 by around 10 fold.

TABLE 16 AUC for Conjugate 10 and Conjugate 10 NP6 Conjugate 10Conjugate 10 NP6 AUC 0-inf (nmol/L*h) 377 3650 Cl (mL/kg/min) 13.0 1.35

Phospho-histone H3 response in NCI-H69 tumors was shown in FIG. 8.Increase in phospho-histone H3 was observed in tumors after treatmentwith Conjugate 10 and Conjugate 10 NP6. At around 50 hour,phospho-histone H3 response for free Conjugate 10 started to decrease,while phospho-histone H3 response for Conjugate 10 NP6 remained high.Pharmacokinetics study suggested a delayed and lengthened response forConjugate 10 NP6.

Therefore, conjugates incorporated in nanoparticles are much moreeffective than the conjugates alone and DM1 alone.

The scope of the present invention is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention can beexcluded from any one or more claims, for any reason, whether or notrelated to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

We claim:
 1. A method of treating lung cancer in a subject in needthereof comprising administering a therapeutically effective amount of aconjugate to the subject, wherein the conjugate comprises an activeagent coupled to a somatostatin receptor (SSTR) targeting moiety by alinker, wherein the active agent is mertansine (DM1) and wherein theSSTR targeting moiety is a peptide, and wherein the conjugate has aformula of

wherein R is selected from the group consisting of H, alkyl, aryl, andamide groups; and Ar₁ and Ar₂ are independently selected from the groupconsisting of heterocyclyl, aryl, and heteroaryl groups.
 2. The methodof claim 1, wherein the molecular weight of the conjugate is less thanabout 50,000 Da, less than about 40,000 Da, less than about 30,000 Da,less than about 20,000 Da, less than about 15,000 Da, less than about10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or lessthan about 3,000 Da.
 3. The method of claim 1, wherein the linker isselected from the group consisting of an ester bond, disulfide, amide,acylhydrazone, ether, carbamate, carbonate, and urea.
 4. The method ofclaim 1, wherein the conjugate is selected from the group consisting ofCompound 57, Compound 35, Compound 37, Compound 39, Compound 41,Compound 43, Compound 45, Compound 47, Compound 49, Compound 51,Compound 55, Compound 59, Compound 61, Compound 63, and Compound
 65. 5.A method of inhibiting the rate of growth of a tumor, the size of atumor or the volume of a tumor, the method comprising contacting thetumor with an effective amount of a conjugate comprising an active agentcoupled to a somatostatin receptor (SSTR) targeting moiety by a linker,wherein the active agent is mertansine (DM1) and wherein the SSTRtargeting moiety is a peptide, wherein the tumor is lung cancer, andwherein the conjugate has a formula of

wherein R is selected from the group consisting of H, alkyl, aryl, andamide groups; and Ar₁ and Ar₂ are independently selected from the groupconsisting of heterocyclyl, aryl, and heteroaryl groups.
 6. A method ofdelivering DM1 to a tumor in a subject, the method comprisingadministering a conjugate to the subject in need thereof, wherein theconjugate comprises an active agent coupled to a somatostatin receptor(SSTR) targeting moiety by a linker, wherein the active agent ismertansine (DM1) and wherein the SSTR targeting moiety is a peptide,wherein the tumor is lung cancer, and wherein the conjugate has aformula of

wherein R is selected from the group consisting of H, alkyl, aryl, andamide groups; and Ar₁ and Ar₂ are independently selected from the groupconsisting of heterocyclyl, aryl, and heteroaryl groups.